Transcriptomic biomarkers for individual risk assessment in new onset heart failure

ABSTRACT

A novel transcriptomic biomarker for prognosis in heart failure has a direct clinical application in prediction of prognosis in new onset heart failure, heart disease, heart disorders and associated heart conditions. This approach should improve individualization of cardiac care and help identify patients at highest risk for circulatory collapse within the first years of presentation with heart failure.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation in part of InternationalApplication No.: PCT/US2008/62281, international filing date May 1,2008, which in turn claims priority to U.S. provisional application No.61/019,749 filed Jan. 8, 2008 and U.S. provisional patent applicationNo. 60/915,224 filed May 1, 2007 which are incorporated herein byreference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with U.S. government support under grant numbersM400-217-2954 and RO-1 HL-65455 both awarded by the National Institutesof Health. The U.S. government may have certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to biomarkers of heart disease novel drugtherapeutic targets, compositions and methods of predicting, diagnosingand treating heart diseases and related disorders thereof. Morespecifically, the invention concerns methods and compositions based onunique molecular signatures associated with various aspects of cardiacdiseases and disorders.

BACKGROUND

The clinical course of patients with newly diagnosed heart failurevaries drastically, with some patients recovering and returning tocompletely normal levels of ejection fraction (EF), while others developsevere symptoms of cardiac decompensation that require insertion of aleft ventricular assist device (LVAD) or a cardiac transplant. Accuraterisk assessment and prediction of prognosis at first presentation arecrucial for appropriate allocation of therapy monitoring and patientmanagement. Prediction tools based on standard criteria have had limitedaccuracy.

SUMMARY

This Summary is provided to present a summary of the invention tobriefly indicate the nature and substance of the invention. It issubmitted with the understanding that it will not be used to interpretor limit the scope or meaning of the claims.

Identifying patients early on in a disease or identifying those at riskof developing cardiovascular diseases and related disorders, wouldprovide both the individual patients and governments with substantialfinancial savings. There is thus a need for accurate prognosticassessment allowing for the adjustment of treatment appropriately andearly enough.

Transcriptomic biomarkers comprise biomolecules and allow for theprediction in the prognostic outcome of heart diseases and disordersthereof. Methods for the prognosis and identification of novel drugtargets are provided.

The transcriptomic biomarkers, methods and assays disclosed herein aredirected to the examination of expression of transcriptomic biomarkersin a mammalian biological sample, e.g. tissue or cell sample, whereinthe determination of that expression of one or more such transcriptomicbiomarkers is predictive of prognostic outcome or diagnostic of cardiacand cardiovascular diseases and disorders, such as for example,myocarditis, Coronary Heart Disease, angina, Acute Coronary Syndrome,Aortic Aneurysm and Dissection, arrhythmias, Cardiomyopathy, CongenitalHeart Disease, congestive heart failure or chronic heart failure,pericarditis, and the like.

In a preferred embodiment a molecular composition comprises gene ornucleic acid sequences: 232669_at (Hypoxia inducible factor 3, alphasubunit), 214951_at (solute carrier family 26, member 10), 243482_at(Epidermal growth factor receptor pathway substrate 15-like 1),226210_s_at (maternally expressed 3), 232159_at (Epidermal growth factorreceptor pathway substrate 15-like 1), 233026_s_at (PDZ domaincontaining), 211996_s_at (KIAA0220-like protein hypothetical gene LOC283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific complementary sequences, fragments, alleles,variants and/or gene products thereof.

In another preferred embodiment, the detection in a cell or patient ofthe biomolecules, complementary sequences, fragments, alleles, variants,derivatives and/or gene products thereof, is prognostic of a goodclinical prognosis in heart failure, biomarkers is predictive ofprognostic outcome or diagnostic of cardiac and cardiovascular diseasesand disorders, such as for example, myocarditis, Coronary Heart Disease,angina, Acute Coronary Syndrome, Aortic Aneurysm and Dissection,arrhythmias, Cardiomyopathy, Congenital Heart Disease, congestive heartfailure or chronic heart failure, pericarditis, and the like.

In another preferred embodiment, the biomolecules, complementarysequences, fragments, alleles, variants, derivatives and/or geneproducts thereof, are modulated or over-expressed at levels by at least1% to a 100% or more in a cell or patient as compared to levels in anormal cell or normal subject.

In another preferred embodiment, the biomolecules, complementarysequences, fragments, alleles, variants, derivatives and/or geneproducts thereof, are modulated or over expressed by about 50% in a cellor a patient as compared to levels in a control, normal cell or normalsubject.

In another preferred embodiment, the biomolecules, complementarysequences, fragments, alleles, variants, derivatives and/or geneproducts thereof, are modulated or over expressed by about 75% in a cellor a patient as compared to levels in a control, normal cell or normalsubject.

In another preferred embodiment, at least ten biomolecules areprognostic in individual risk assessment in a patient for onset of heartfailure, cardiac and cardiovascular diseases and disorders, such as forexample, myocarditis, Coronary Heart Disease, angina, Acute CoronarySyndrome, Aortic Aneurysm and Dissection, arrhythmias, Cardiomyopathy,Congenital Heart Disease, congestive heart failure or chronic heartfailure, pericarditis, and the like.

In another preferred embodiment, a plurality of biomolecules areprognostic in individual risk assessment in a patient for onset of heartfailure and predictive of prognostic outcome or diagnostic of cardiacand cardiovascular diseases and disorders, such as for example,myocarditis, Coronary Heart Disease, angina, Acute Coronary Syndrome,Aortic Aneurysm and Dissection, arrhythmias, Cardiomyopathy, CongenitalHeart Disease, congestive heart failure or chronic heart failure,pericarditis, and the like.

In another preferred embodiment, a biomarker (TBB) for predicting aprognosis of heart failure, comprising nucleic acidsequences/biomolecules comprising: 232669_at (Hypoxia inducible factor3, alpha subunit), 214951_at (solute carrier family 26, member 10),243482_at (Epidermal growth factor receptor pathway substrate 15-like1), 226210_s_at (maternally expressed 3), 232159_at (Epidermal growthfactor receptor pathway substrate 15-like 1), 233026_s_at (PDZ domaincontaining), 211996_s_at (KIAA0220-like protein hypothetical gene LOC283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, derivatives, variants and/or gene products thereof.

In another preferred embodiment, the biomolecules of the biomarkercomprising biomolecules, complementary sequences, fragments, alleles,derivatives, variants and gene products thereof, are modulated and/orover-expressed at levels by at least 1% to a 100% or more in a cell orpatient as compared to levels in a normal cell or normal subject.

In another preferred embodiment, the biomolecules of the biomarkercomprising biomolecules, complementary sequences, fragments, alleles,derivatives, variants and gene products thereof, comprise a molecularsignature wherein the biomolecules are modulated with respect to eachother and normal controls are prognostic of increased risk of disease orthe outcome of a disease. Thus, one or more biomolecules which comprisea molecular signature or expression profile for a specific disease maybe up-regulated or down-regulated in relation to each other.

In another preferred embodiment, the nucleic acid sequences,complementary sequences, fragments, alleles, variants, derivativesand/or gene products thereof, are modulated and/or over-expressed byabout 50% in a cell or a patient as compared to levels in a normal cellor normal subject.

In another preferred embodiment, the nucleic acid sequences,complementary sequences, fragments, alleles, variants, derivativesand/or gene products thereof, are modulated and/or over-expressed byabout 75% in a cell or a patient as compared to levels in a normal cellor normal subject.

In another preferred embodiment, an antibody or aptamer specific foreach gene sequence comprising: 232669_at (Hypoxia inducible factor 3,alpha subunit), 214951_at (solute carrier family 26, member 10),243482_at (Epidermal growth factor receptor pathway substrate 15-like1), 226210_s_at (maternally expressed 3), 232159_at (Epidermal growthfactor receptor pathway substrate 15-like 1), 233026_s_at (PDZ domaincontaining), 211996_s_at (KIAA0220-like protein hypothetical gene LOC283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, derivatives, variants and/or gene products thereof.

In another preferred embodiment, a biochip comprises nucleic acidsequences: 232669_at (Hypoxia inducible factor 3, alpha subunit),214951_at (solute carrier family 26, member 10), 243482_at (Epidermalgrowth factor receptor pathway substrate 15-like 1), 226210_s_at(maternally expressed 3), 232159_at (Epidermal growth factor receptorpathway substrate 15-like 1), 233026_s_at (PDZ domain containing),211996_s_at (KIAA0220-like protein hypothetical gene LOC 283846),243774_at (mucin 20, cell surface associated), 242551_at (Chromosome 18open reading frame), 244548_at (Rho GTPase activating protein 26),244208_at (Checkpoint suppressor 1), 239984_at (Sodium channel,voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis, cloneADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at (protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, derivatives, variants and/or gene products thereof.

In one embodiment, the biochip comprises at least ten nucleic acidsequences, complementary sequences, fragments, alleles, variants,derivatives and/or gene products thereof.

In a preferred embodiment, a method of assessing identifying anddistinguishing between patients at a high risk of heart disease andpatients with a good prognosis for recovery comprising: identifying in abiological sample from a patient a molecular signature comprising atranscriptomic based biomarker (TBB): 232669_at (Hypoxia induciblefactor 3, alpha subunit), 214951_at (solute carrier family 26, member10), 243482_at (Epidermal growth factor receptor pathway substrate15-like 1), 226210_s_at (maternally expressed 3), 232159_at (Epidermalgrowth factor receptor pathway substrate 15-like 1), 233026_s_at (PDZdomain containing), 211996_s_at (KIAA0220-like protein hypothetical geneLOC 283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, derivatives, variants and/or gene products thereof; assessingthe probability of identification of each component gene in each sample;assigning each to a class; and, differentiating between idiopathiccardiomyopathy and myocarditis.

In another preferred embodiment, the transcriptomic biomarker comprisesan expression profile or molecular signature of the biomolecules. Forexample, the expression profile of each biomolecule with respect to eachother and/or to controls is prognostic or diagnostic of a disease. Forexample, in some aspects, some of the biomolecules are up-regulated,down-regulated or not expressed relative to each other. This is amolecular signature which would be diagnostic or prognostic, riskassessment etc of a specific disease.

In a preferred embodiment, a method of assessing identifying anddistinguishing between patients at a high risk of heart disease andpatients with a good prognosis for recovery comprising: identifying in abiological sample from a patient a molecular signature comprising atranscriptomic based biomarker (TBB): 232669_at (Hypoxia induciblefactor 3, alpha subunit), 214951_at (solute carrier family 26, member10), 243482_at (Epidermal growth factor receptor pathway substrate15-like 1), 226210_s_at (maternally expressed 3), 232159_at (Epidermalgrowth factor receptor pathway substrate 15-like 1), 233026_s_at (PDZdomain containing), 211996_s_at (KIAA0220-like protein hypothetical geneLOC 283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), and 201510_at E74-like factor 3 (ets domaintranscription factor, epithelial specific).

In a preferred embodiment, the biomarker is identified from a patient byisolating nucleic acids obtained from a biological sample.

In another preferred embodiment, the nucleic acids are hybridized to thebiochip and raw intensity values from microarray hybridization arenormalized and phenotype specific differences in gene expression areidentified. The differences in gene expression are identified bysignificance analysis of microarrays, wherein significance is definedwith a q-value and multiple comparisons comprise an adjusted p-value.

In another preferred embodiment, the phenotype specificity is identifiedby creating a classifier in a training set comprising about 66% of dataobtained, with subsequent validation in a test set comprising about 33%of data obtained and defining a phenotype specific nearest shrunkencentroid for classification. The phenotype specific nearest shrunkencentroid comprises balancing about a 10-fold cross validation in atraining set.

In another preferred embodiment, a method of predicting a prognosticoutcome for heart disease or recovery from heart failure comprising:identifying in a biological sample from a patient a compositioncomprising biomoelcules: 232669_at (Hypoxia inducible factor 3, alphasubunit), 214951_at (solute carrier family 26, member 10), 243482_at(Epidermal growth factor receptor pathway substrate 15-like 1),226210_s_at (maternally expressed 3), 232159_at (Epidermal growth factorreceptor pathway substrate 15-like 1), 233026_s_at (PDZ domaincontaining), 211996_s_at (KIAA0220-like protein hypothetical gene LOC283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, derivatives, variants and/or gene products thereof; and,assessing the probability of identification of each component gene ineach sample; assigning each to a class; and, predicting the prognosticoutcome for heart disease or recovery from heart failure.

In one embodiment, the biomolecules are over-expressed by at least about5% as compared to a normal cell or normal subject.

In another preferred embodiment, a method of identifying anddistinguishing between patients at a high risk of heart disease andpatients with a good prognosis for recovery comprising: identifyingbiomolecules comprising: 232669_at (Hypoxia inducible factor 3, alphasubunit), 214951_at (solute carrier family 26, member 10), 243482_at(Epidermal growth factor receptor pathway substrate 15-like 1),226210_s_at (maternally expressed 3), 232159_at (Epidermal growth factorreceptor pathway substrate 15-like 1), 233026_s_at (PDZ domaincontaining), 211996_s_at (KIAA0220-like protein hypothetical gene LOC283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, derivatives, variants and/or gene products thereof; assessingthe probability of identification of each component gene in each sample;assigning each to a class; and, identifying and distinguishing betweenpatients at a high risk of heart disease and patients with a goodprognosis for recovery.

In another preferred embodiment, a molecular composition comprisingbiomolecules: 1558458_at (Hypothetical LOC401320), 1560049_at (CUGtriplet repeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNA bindingmotif protein 5, RBM5), 201655_s_at (heparan sulfate proteoglycan 2(perlecan), HSPG2), 202379_s_at (natural killer-tumor recognitionsequence, NKTR), 202808_at, 203071_at (sema domain, immunoglobulindomain (Ig), short basic domain, secreted, (semaphorin) 3B, SEMA3B),203748_x_at (RNA binding motif, single stranded interacting protein 1,RBMS1), 203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4,ABCD4), 204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7,MYH6///MYH7), 204978_at (splicing factor, arginine/serine-rich 16,SFRS16), 206209_s_at (carbonic anhydrase IV, CA4), 207541_s_at (exosomecomponent 10, EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at(cysteine-rich protein 2, CRIP2), 209354_at (tumor necrosis factorreceptor superfamily, member 14 TNFRSF14), 210628_x_at (latenttransforming growth factor beta binding protein 4, LTBP4), 211909_x_at(prostaglandin E receptor 3 (subtype EP3), PTGER3), 211996_at(KIAA0220-like protein, nuclear pore complex (LOC23117), 212487_at (Gpatch domain containing 8, GPATCH8), 213946_s_at (obscurin-like 1,OBSL1), 214951_at (solute carrier family 26, member 10, SLC26A10),220219_s_at (leucine rich repeat containing 37A, LRRC37A), 221071_at,221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27),221806_s_at (SET domain containing 5, SETD5), 221833_at (Lon peptidase2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S. cerevisiae), LUC7L),224260_at (CDNA clone IMAGE:4478733), 225562_at (AS p21 proteinactivator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156 (from cloneDKFZp762N156), 227968_at (Parkinson disease 7 domain containing 1,PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9, MRPS9),229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis, cloneADKA03430), 238185_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 241597_at (Arginine-glutamic acid dipeptide (RE)repeats, RERE), 242551_at (Chromosome 18 open reading frame 1, C18orf1),244208_at (Checkpoint suppressor 1, CHES1), 244494_at (Zinc finger,DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPase activatingprotein 26, ARHGAP26) complementary sequences, fragments, derivatives,alleles, variants and/or gene products thereof.

Preferably, the detection in a cell or patient of the biomolecules,complementary sequences, fragments, alleles, derivatives, variants andgene products thereof, is predictive of clinical diagnostic outcome andprognosis of heart failure, predictive of prognostic outcome ordiagnostic of cardiac and cardiovascular diseases and disorders, such asfor example, myocarditis, Coronary Heart Disease, angina, Acute CoronarySyndrome, Aortic Aneurysm and Dissection, arrhythmias, Cardiomyopathy,Congenital Heart Disease, congestive heart failure or chronic heartfailure, pericarditis, and the like.

Preferably, the biomolecules, complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof, aremodulated and/or over-expressed at levels by at least 1% to a 100%,200%, 300% or more in a cell or patient as compared to levels in anormal cell or normal subject.

Preferably, the biomolecules, complementary sequences, fragments,alleles, derivatives, variants and gene products thereof, produce amolecular signature or expression profile, wherein one or morebiomolecules are differentially expressed to each other and to normalcontrols.

In an alternative embodiment, at least nine biomolecules are prognosticin individual risk assessment in a patient, predictive of clinicaldiagnostic outcome and prognosis of heart failure.

In another preferred embodiment, a plurality of gene sequences areprognostic in individual risk assessment in a patient for onset of heartfailure.

In another preferred embodiment, a biomarker (TBB) predictive ofclinical diagnostic outcome and prognosis of heart failure, comprisingnucleic acid sequences/biomolecules comprising: 1558458_at (HypotheticalLOC401320), 1560049_at (CUG triplet repeat, RNA binding protein 2,CUGBP2), 201394_s_at (RNA binding motif protein 5, RBM5), 201655_s_at(heparan sulfate proteoglycan 2 (perlecan), HSPG2), 202379_s_at (naturalkiller-tumor recognition sequence, NKTR), 202808_at, 203071_at (semadomain, immunoglobulin domain (Ig), short basic domain, secreted,(semaphorin) 3B, SEMA3B), 203748_x_at (RNA binding motif, singlestranded interacting protein 1, RBMS1), 203981_s_at (ATP-bindingcassette, sub-family D (ALD), member 4, ABCD4), 204737_s_at (myosin,heavy chain 6, myosin, heavy chain 7, MYH6///MYH7), 204978_at (splicingfactor, arginine/serine-rich 16, SFRS16), 206209_s_at (carbonicanhydrase IV, CA4), 207541_s_at (exosome component 10, EXOSC10),207798_s_at (ataxin 2-like, ATXN2L), 208978_at (cysteine-rich protein 2,CRIP2), 209354_at (tumor necrosis factor receptor superfamily, member 14TNFRSF14), 210628_x_at (latent transforming growth factor beta bindingprotein 4, LTBP4), 211909_x_at (prostaglandin E receptor 3 (subtypeEP3), PTGER3), 211996_at (KIAA0220-like protein, nuclear pore complex(LOC23117), 212487_at (G patch domain containing 8, GPATCH8),213946_s_at (obscurin-like 1, OBSL1), 214951_at (solute carrier family26, member 10, SLC26A10), 220219_s_at (leucine rich repeat containing37A, LRRC37A), 221071_at, 221780_s_at (DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 27DDX27), 221806_s_at (SET domain containing 5, SETD5),221833_at (Lon peptidase 2, peroxisomal, LONP2), 223546_x_at (LUC7-like(S. cerevisiae), LUC7L), 224260_at (CDNA clone IMAGE:4478733), 225562_at(AS p21 protein activator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156(from clone DKFZp762N156), 227968_at (Parkinson disease 7 domaincontaining 1, PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9,MRPS9), 229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis,clone ADKA03430), 238185_at (RNA binding motif, single strandedinteracting protein 1, RBMS1), 241597_at (Arginine-glutamic aciddipeptide (RE) repeats, RERE), 242551_at (Chromosome 18 open readingframe 1, C18orf1), 0.244208_at (Checkpoint suppressor 1, CHES1),244494_at (Zinc finger, DHHC-type containing 1, ZDHHC1), and 244548_at(Rho GTPase activating protein 26, ARHGAP26) complementary sequences,fragments, alleles, derivatives, variants and/or gene products thereof.

In one embodiment, detection in a cell or patient of the gene sequences,complementary sequences, fragments, alleles, derivatives, variants andgene products thereof, is predictive of clinical diagnostic outcome andprognosis of heart failure, and is predictive of prognostic outcome ordiagnostic of cardiac and cardiovascular diseases and disorders, such asfor example, myocarditis, Coronary Heart Disease, angina, Acute CoronarySyndrome, Aortic Aneurysm and Dissection, arrhythmias, Cardiomyopathy,Congenital Heart Disease, congestive heart failure or chronic heartfailure, pericarditis, and the like.

In another a biochip comprises nucleic acid sequences: 1558458_at(Hypothetical LOC401320), 1560049_at (CUG triplet repeat, RNA bindingprotein 2, CUGBP2), 201394_s_at (RNA binding motif protein 5, RBM5),201655_s_at (heparan sulfate proteoglycan 2 (perlecan), HSPG2),202379_s_at (natural killer-tumor recognition sequence, NKTR),202808_at, 203071_at (sema domain, immunoglobulin domain (Ig), shortbasic domain, secreted, (semaphorin) 3B, SEMA3B), 203748_x_at (RNAbinding motif, single stranded interacting protein 1, RBMS1),203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4, ABCD4),204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7, MYH6///MYH7),204978_at (splicing factor, arginine/serine-rich 16, SFRS16),206209_s_at (carbonic anhydrase IV, CA4), 207541_s_at (exosome component10, EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at(cysteine-rich protein 2, CRIP2), 209354_at (tumor necrosis factorreceptor superfamily, member 14 TNFRSF14), 210628_x_at (latenttransforming growth factor beta binding protein 4, LTBP4), 211909_x_at(prostaglandin E receptor 3 (subtype EP3), PTGER3), 211996_at(KIAA0220-like protein, nuclear pore complex (LOC23117), 212487_at (Gpatch domain containing 8, GPATCH8), 213946_s_at (obscurin-like 1,OBSL1), 214951_at (solute carrier family 26, member 10, SLC26A10),220219_s_at (leucine rich repeat containing 37A, LRRC37A), 221071_at,221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27),221806_s_at (SET domain containing 5, SETD5), 221833_at (Lon peptidase2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S. cerevisiae), LUC7L),224260_at (CDNA clone IMAGE:4478733), 225562_at (AS p21 proteinactivator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156 (from cloneDKFZp762N156), 227968_at (Parkinson disease 7 domain containing 1,PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9, MRPS9),229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis, cloneADKA03430), 238185_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 241597_at (Arginine-glutamic acid dipeptide (RE)repeats, RERE), 242551_at (Chromosome 18 open reading frame 1, C18orf1),244208_at (Checkpoint suppressor 1, CHES1), 244494_at (Zinc finger,DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPase activatingprotein 26, ARHGAP26), complementary sequences, fragments, alleles,derivatives, variants and/or gene products thereof.

In one embodiment, the biochip comprises at least ten nucleic acidsequences, complementary sequences, fragments, alleles, variants,derivatives and/or gene products thereof.

In another preferred embodiment, an antibody or aptamer is specific foreach of: 1558458_at (Hypothetical LOC401320), 1560049_at (CUG tripletrepeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNA binding motifprotein 5, RBM5), 201655_s_at (heparan sulfate proteoglycan 2(perlecan), HSPG2), 202379_s_at (natural killer-tumor recognitionsequence, NKTR), 202808_at, 203071_at (sema domain, immunoglobulindomain (Ig), short basic domain, secreted, (semaphorin) 3B, SEMA3B),203748_x_at (RNA binding motif, single stranded interacting protein 1,RBMS1), 203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4,ABCD4), 204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7,MYH6///MYH7), 204978_at (splicing factor, arginine/serine-rich 16,SFRS16), 206209_s_at (carbonic anhydrase IV, CA4), 207541_s_at (exosomecomponent 10, EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at(cysteine-rich protein 2, CRIP2), 209354_at (tumor necrosis factorreceptor superfamily, member 14 TNFRSF14), 210628_x_at (latenttransforming growth factor beta binding protein 4, LTBP4), 211909_x_at(prostaglandin E receptor 3 (subtype EP3), PTGER3), 211996_at(KIAA0220-like protein, nuclear pore complex (LOC23117), 212487_at (Gpatch domain containing 8, GPATCH8), 213946_s_at (obscurin-like 1,OBSL1), 214951_at (solute carrier family 26, member 10, SLC26A10),220219_s_at (leucine rich repeat containing 37A, LRRC37A), 221071_at,221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27),221806_s_at (SET domain containing 5, SETD5), 221833_at (Lon peptidase2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S. cerevisiae), LUC7L),224260_at (CDNA clone IMAGE:4478733), 225562_at (AS p21 proteinactivator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156 (from cloneDKFZp762N156), 227968_at (Parkinson disease 7 domain containing 1,PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9, MRPS9),229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis, cloneADKA03430), 238185_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 241597_at (Arginine-glutamic acid dipeptide (RE)repeats, RERE), 242551_at (Chromosome 18 open reading frame 1, C18orf1),244208_at (Checkpoint suppressor 1, CHES1), 244494_at (Zinc finger,DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPase activatingprotein 26, ARHGAP26) complementary sequences, fragments, derivatives,alleles, variants and/or gene products thereof.

In another preferred embodiment, a method of assessing, identifying anddistinguishing between patients at a high risk of heart disease andpatients with a good prognosis for recovery comprises identifying in abiological sample from a patient a molecular signature comprising atranscriptomic based biomarker (TBB): 1558458_at (HypotheticalLOC401320), 1560049_at (CUG triplet repeat, RNA binding protein 2,CUGBP2), 201394_s_at (RNA binding motif protein 5, RBM5), 201655_s_at(heparan sulfate proteoglycan 2 (perlecan), HSPG2), 202379_s_at (naturalkiller-tumor recognition sequence, NKTR), 202808_at, 203071_at (semadomain, immunoglobulin domain (Ig), short basic domain, secreted,(semaphorin) 3B, SEMA3B), 203748_x_at (RNA binding motif, singlestranded interacting protein 1, RBMS1), 203981_s_at (ATP-bindingcassette, sub-family D (ALD), member 4, ABCD4), 204737_s_at (myosin,heavy chain 6, myosin, heavy chain 7, MYH6///MYH7), 204978_at (splicingfactor, arginine/serine-rich 16, SFRS16), 206209_s_at (carbonicanhydrase IV, CA4), 207541_s_at (exosome component 10, EXOSC10),207798_s_at (ataxin 2-like, ATXN2L), 208978_at (cysteine-rich protein 2,CRIP2), 209354_at (tumor necrosis factor receptor superfamily, member 14TNFRSF14), 210628_x_at (latent transforming growth factor beta bindingprotein 4, LTBP4), 211909_x_at (prostaglandin E receptor 3 (subtypeEP3), PTGER3), 211996_at (KIAA0220-like protein, nuclear pore complex(LOC23117), 212487_at (G patch domain containing 8, GPATCH8),213946_s_at (obscurin-like 1, OBSL1), 214951_at (solute carrier family26, member 10, SLC26A10), 220219_s_at (leucine rich repeat containing37A, LRRC37A), 221071_at, 221780_s_at (DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 27DDX27), 221806_s_at (SET domain containing 5, SETD5),221833_at (Lon peptidase 2, peroxisomal, LONP2), 223546_x_at (LUC7-like(S. cerevisiae), LUC7L), 224260_at (CDNA clone IMAGE:4478733), 225562_at(AS p21 protein activator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156(from clone DKFZp762N156), 227968_at (Parkinson disease 7 domaincontaining 1, PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9,MRPS9), 229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis,clone ADKA03430), 238185_at (RNA binding motif, single strandedinteracting protein 1, RBMS1), 241597_at (Arginine-glutamic aciddipeptide (RE) repeats, RERE), 242551_at (Chromosome 18 open readingframe 1, C18orf1), 244208_at (Checkpoint suppressor 1, CHES1), 244494_at(Zinc finger, DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPaseactivating protein 26, ARHGAP26) complementary sequences, fragments,alleles, derivatives, variants and gene products thereof; assessing theprobability of identification of each component gene in each sample;assigning each to a class; and, differentiating between idiopathiccardiomyopathy and myocarditis.

In another preferred embodiment, a method of predicting a prognosticoutcome for heart disease or recovery from heart failure comprisesidentifying in a biological sample from a patient a compositioncomprising gene sequences: 1558458_at (Hypothetical LOC401320),1560049_at (CUG triplet repeat, RNA binding protein 2, CUGBP2),201394_s_at (RNA binding motif protein 5, RBM5), 201655_s_at (heparansulfate proteoglycan 2 (perlecan), HSPG2), 202379_s_at (naturalkiller-tumor recognition sequence, NKTR), 202808_at, 203071_at (semadomain, immunoglobulin domain (Ig), short basic domain, secreted,(semaphorin) 3B, SEMA3B), 203748_x_at (RNA binding motif, singlestranded interacting protein 1, RBMS1), 203981_s_at (ATP-bindingcassette, sub-family D (ALD), member 4, ABCD4), 204737_s_at (myosin,heavy chain 6, myosin, heavy chain 7, MYH6///MYH7), 204978_at (splicingfactor, arginine/serine-rich 16, SFRS16), 206209_s_at (carbonicanhydrase IV, CA4), 207541_s_at (exosome component 10, EXOSC10),207798_s_at (ataxin 2-like, ATXN2L), 208978_at (cysteine-rich protein 2,CRIP2), 209354_at (tumor necrosis factor receptor superfamily, member 14TNFRSF14), 210628_x_at (latent transforming growth factor beta bindingprotein 4, LTBP4), 211909_x_at (prostaglandin E receptor 3 (subtypeEP3), PTGER3), 211996_at (KIAA0220-like protein, nuclear pore complex(LOC23117), 212487_at (G patch domain containing 8, GPATCH8),213946_s_at (obscurin-like 1, OBSL1), 214951_at (solute carrier family26, member 10, SLC26A10), 220219_s_at (leucine rich repeat containing37A, LRRC37A), 221071_at, 221780_s_at (DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 27DDX27), 221806_s_at (SET domain containing 5, SETD5),221833_at (Lon peptidase 2, peroxisomal, LONP2), 223546_x_at (LUC7-like(S. cerevisiae), LUC7L), 224260_at (CDNA clone IMAGE:4478733), 225562_at(AS p21 protein activator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156(from clone DKFZp762N156), 227968_at (Parkinson disease 7 domaincontaining 1, PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9,MRPS9), 229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis,clone ADKA03430), 238185_at (RNA binding motif, single strandedinteracting protein 1, RBMS1), 241597_at (Arginine-glutamic aciddipeptide (RE) repeats, RERE), 242551_at (Chromosome 18 open readingframe 1, C18orf1), 244208_at (Checkpoint suppressor 1, CHES1), 244494_at(Zinc finger, DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPaseactivating protein 26, ARHGAP26), complementary sequences, fragments,alleles, derivatives, variants and/or gene products thereof; and,assessing the probability of identification of each component gene ineach sample; assigning each to a class; and, predicting heart disease orcardiomyopathy.

In another preferred embodiment, a method of identifying anddistinguishing between patients at a high risk of heart disease andpatients with a good prognosis for recovery comprises identifying genesequences comprising: 1558458_at (Hypothetical LOC401320), 1560049_at(CUG triplet repeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNAbinding motif protein 5, RBM5), 201655_s_at (heparan sulfateproteoglycan 2 (perlecan), HSPG2), 202379_s_at (natural killer-tumorrecognition sequence, NKTR), 202808_at, 203071_at (sema domain,immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin)3B, SEMA3B), 203748_x _at (RNA binding motif, single strandedinteracting protein 1, RBMS1), 203981_s_at (ATP-binding cassette,sub-family D (ALD), member 4, ABCD4), 204737_s_at (myosin, heavy chain6, myosin, heavy chain 7, MYH6///MYH7), 204978_at (splicing factor,arginine/serine-rich 16, SFRS16), 206209_s_at (carbonic anhydrase IV,CA4), 207541_s_at (exosome component 10, EXOSC10), 207798_s_at (ataxin2-like, ATXN2L), 208978_at (cysteine-rich protein 2, CRIP2), 209354_at(tumor necrosis factor receptor superfamily, member 14 TNFRSF14),210628_x_at (latent transforming growth factor beta binding protein 4,LTBP4), 211909_x_at (prostaglandin E receptor 3 (subtype EP3), PTGER3),211996_at (KIAA0220-like protein, nuclear pore complex (LOC23117),212487_at (G patch domain containing 8, GPATCH8), 213946_s_at(obscurin-like 1, OBSL1), 214951_at (solute carrier family 26, member10, SLC26A10), 220219_s_at (leucine rich repeat containing 37A,LRRC37A), 221071_at, 221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide27DDX27), 221806_s_at (SET domain containing 5, SETD5), 221833_at (Lonpeptidase 2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S.cerevisiae), LUC7L), 224260_at (CDNA clone IMAGE:4478733), 225562_at (ASp21 protein activator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156(from clone DKFZp762N156), 227968_at (Parkinson disease 7 domaincontaining 1, PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9,MRPS9), 229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis,clone ADKA03430), 238185_at (RNA binding motif, single strandedinteracting protein 1, RBMS1), 241597_at (Arginine-glutamic aciddipeptide (RE) repeats, RERE), 242551_at (Chromosome 18 open readingframe 1, C18orf1), 244208_at (Checkpoint suppressor 1, CHES1), 244494_at(Zinc finger, DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPaseactivating protein 26, ARHGAP26) complementary sequences, fragments,alleles, derivatives, variants and/or gene products thereof; assessingthe probability of identification of each component gene in each sample;assigning each to a class; and, identifying and distinguishing betweenpatients at a high risk of heart disease and patients with a goodprognosis for recovery.

In another preferred embodiment, a cell expressing any one or more ofnucleic acid sequences or products thereof: 232669_at (Hypoxia induciblefactor 3, alpha subunit), 214951_at (solute carrier family 26, member10), 243482_at (Epidermal growth factor receptor pathway substrate15-like 1), 226210_s_at (maternally expressed 3), 232159_at (Epidermalgrowth factor receptor pathway substrate 15-like 1), 233026_s_at (PDZdomain containing), 211996_s_at (KIAA0220-like protein hypothetical geneLOC 283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific).

In another preferred embodiment, a vector expressing any one or morenucleic acid products comprising: 232669_at (Hypoxia inducible factor 3,alpha subunit), 214951_at (solute carrier family 26, member 10),243482_at (Epidermal growth factor receptor pathway substrate 15-like1), 226210_s_at (maternally expressed 3), 232159_at (Epidermal growthfactor receptor pathway substrate 15-like 1), 233026_s_at (PDZ domaincontaining), 211996_s_at (KIAA0220-like protein hypothetical gene LOC283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific) or combinations thereof.

In another preferred embodiment, a cell expresses any one or more ofnucleic acid sequences or products thereof comprising: 1558458_at(Hypothetical LOC401320), 1560049_at (CUG triplet repeat, RNA bindingprotein 2, CUGBP2), 201394_s_at (RNA binding motif protein 5, RBM5),201655_s_at (heparan sulfate proteoglycan 2 (perlecan), HSPG2),202379_s_at (natural killer-tumor recognition sequence, NKTR),202808_at, 203071_at (sema domain, immunoglobulin domain (Ig), shortbasic domain, secreted, (semaphorin) 3B, SEMA3B), 203748_x_at (RNAbinding motif, single stranded interacting protein 1, RBMS1),203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4, ABCD4),204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7, MYH6///MYH7),204978_at (splicing factor, arginine/serine-rich 16, SFRS16),206209_s_at (carbonic anhydrase IV, CA4), 207541_s_at (exosome component10, EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at(cysteine-rich protein 2, CRIP2), 209354_at (tumor necrosis factorreceptor superfamily, member 14 TNFRSF14), 210628_x_at (latenttransforming growth factor beta binding protein 4, LTBP4), 211909_x_at(prostaglandin E receptor 3 (subtype EP3), PTGER3), 211996_at(KIAA0220-like protein, nuclear pore complex (LOC23117), 212487_at (Gpatch domain containing 8, GPATCH8), 213946_s_at (obscurin-like 1,OBSL1), 214951_at (solute carrier family 26, member 10, SLC26A10),220219_s_at (leucine rich repeat containing 37A, LRRC37A), 221071_at,221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27),221806_s_at (SET domain containing 5, SETD5), 221833_at (Lon peptidase2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S. cerevisiae), LUC7L),224260_at (CDNA clone IMAGE:4478733), 225562_at (AS p21 proteinactivator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156 (from cloneDKFZp762N156), 227968_at (Parkinson disease 7 domain containing 1,PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9, MRPS9),229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis, cloneADKA03430), 238185_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 241597_at (Arginine-glutamic acid dipeptide (RE)repeats, RERE), 242551_at (Chromosome 18 open reading frame 1, C18orf1),244208_at (Checkpoint suppressor 1, CHES1), 244494_at (Zinc finger,DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPase activatingprotein 26, ARHGAP26) complementary sequences, fragments, alleles,derivatives, variants and/or gene products thereof.

In another preferred embodiment, a vector expresses any one or more ofnucleic acid sequences comprising: 1558458_at (Hypothetical LOC401320),1560049_at (CUG triplet repeat, RNA binding protein 2, CUGBP2),201394_s_at (RNA binding motif protein 5, RBM5), 201655_s_at (heparansulfate proteoglycan 2 (perlecan), HSPG2), 202379_s_at (naturalkiller-tumor recognition sequence, NKTR), 202808_at, 203071_at (semadomain, immunoglobulin domain (Ig), short basic domain, secreted,(semaphorin) 3B, SEMA3B), 203748_x_at (RNA binding motif, singlestranded interacting protein 1, RBMS1), 203981_s_at (ATP-bindingcassette, sub-family D (ALD), member 4, ABCD4), 204737_s_at (myosin,heavy chain 6, myosin, heavy chain 7, MYH6///MYH7), 204978_at (splicingfactor, arginine/serine-rich 16, SFRS16), 206209_s_at (carbonicanhydrase IV, CA4), 207541_s_at (exosome component 10, EXOSC10),207798_s_at (ataxin 2-like, ATXN2L), 208978_at (cysteine-rich protein 2,CRIP2), 209354_at (tumor necrosis factor receptor superfamily, member 14TNFRSF14), 210628_x_at (latent transforming growth factor beta bindingprotein 4, LTBP4), 211909_x_at (prostaglandin E receptor 3 (subtypeEP3), PTGER3), 211996_at (KIAA0220-like protein, nuclear pore complex(LOC23117), 212487_at (G patch domain containing 8, GPATCH8),213946_s_at (obscurin-like 1, OBSL1), 214951_at (solute carrier family26, member 10, SLC26A10), 220219_s_at (leucine rich repeat containing37A, LRRC37A), 221071_at, 221780_s_at (DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 27DDX27), 221806_s_at (SET domain containing 5, SETD5),221833_at (Lon peptidase 2, peroxisomal, LONP2), 223546_x_at (LUC7-like(S. cerevisiae), LUC7L), 224260_at (CDNA clone IMAGE:4478733), 225562_at(AS p21 protein activator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156(from clone DKFZp762N156), 227968_at (Parkinson disease 7 domaincontaining 1, PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9,MRPS9), 229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis,clone ADKA03430), 238185_at (RNA binding motif, single strandedinteracting protein 1, RBMS1), 241597_at (Arginine-glutamic aciddipeptide (RE) repeats, RERE), 242551_at (Chromosome 18 open readingframe 1, C18orf1), 244208_at (Checkpoint suppressor 1, CHES1), 244494_at(Zinc finger, DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPaseactivating protein 26, ARHGAP26) complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof.

Other aspects of the invention are described infra.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is pointed out with particularity in the appended claims.The above and further advantages of this invention may be betterunderstood by referring to the following description taken inconjunction with the accompanying drawings, in which:

FIG. 1 is a scan of a photograph showing an analysis of extracted totalRNA with Agilent 2100 Bioanalyzer. Every sample was tested for itsintegrity and purity before microarray hybridization. The photographdepicts a gel of 12 samples with consistent bands of 18S and 28S RNA.The left lane contains the reference marker.

FIG. 2 is a schematic representation of train and test sets as used forthe development of a classifier with Prediction Analysis of Microarrays(PAM). All samples obtained from patients with poor prognosis (PP, n=18)were selected from a biorepository (n=180) and those with good prognosis(GP, n=25) were chosen in a case-control fashion (see text fordefinitions of PP and GP). The classifier, a nearest shrunken centroid,was developed in ⅔ of the data (17 samples with GP; 12 samples with PP)with subsequent validation in the remaining ⅓ of data (8 samples withGP; 6 samples with PP). The overall test accuracy of the TBB wascalculated from 50 random partitions into train and test sets.

FIG. 3 is a heatmap of samples from all patients with idiopathic dilatedcardiomyopathy (n=43). Each column corresponds to a patient sample andeach row represents a gene. Samples classified as having PP form adistinct cluster and are highlighted in a red square. Downregulatedgenes are depicted with red, whereas upregulated genes are labeled blue.Yellow arrows denote misclassified samples.

FIG. 4 is a pie chart illustrating involved pathways within theprognostic biomarker. Major pathways overexpressed in GP patientsincluded transcription (26%), protein binding (15%), ion transport (13%)and neuromuscular development (10%).

FIG. 5 shows two graphs illustrating the Functional improvement ofEjection fraction (EF) from baseline to endpoint: Within all enrolledcases of idiopathic cardiomyopathy (n=43), we further analyzed those ofwhom echocardiographic measurements at baseline and endpoint wereavailable (n=17). Samples are classified into GP or PP based upon TBBprediction. Patients classified as GP (n=11, average follow-up: 49.9±21mo) experienced improvement of EF (*P=0.0009), while PP (n=6, averagefollow-up: 6.2±2.9 mo) did not. Red line depicts one misclassifiedsample. Error bars represent SEM.

FIG. 6 is a graph showing the crossvalidation of the training set:starting from a set of 9 genes, increasing the threshold leads to aconstant increase of the misclassification error after a limit of 2.1

FIG. 7 is a graph showing crossvalidation of the training set:Increasing the threshold constantly increases the misclassificationerror after a limit of 2.1

FIG. 8 is a heatmap using 9 genes signature: this heatmap was created byan unsupervised clustering approach using standardization by mean levelsof expression. Each column represents a sample and each line correspondsto a gene, for which the IDs from affymetrix are listed on the rightside. For further details about the gene annotations see table 5. A redcolor means low expression of the gene, whereas a blue colordemonstrates high gene expression levels. Three samples from patientswith bad prognosis were grouped wrong (encircled).

FIG. 9 shows a heatmap using 43 genes signature: this heatmap wascreated with the same unsupervised hierarchical clustering algorithm.Only one sample was classified wrong (BP-10). The history of thispatient, it was later found out, was that he was first diagnosed withheart failure more than 5 years before he died, which suggests that hewas actually a long term survivor and recognized correctly by ouralgorithm. In this study, he was categorized as a patient with poorprognosis, because the biopsy was obtained one year before he died.

FIG. 10 is a graph showing the nearest shrunken centroid of the “9 genesclassifier”: The centroids were calculated in PAM from the averageexpression for each gene in each class divided by the within-classstandard deviation for that gene. The nearest shrunken centroidclassification “shrinks” each of the class centroids toward the overallcentroid for all classes by the threshold. Nearest centroidclassification takes the gene expression profile of a new sample, andcompares it to each of these class centroids. The class whose centroidthat it is closest to, in squared distance, is the predicted class forthat new sample. Each line in the graph represents a gene. The redcentroid characterizes group 1 (bad prognosis), the green centroidcharacterizes group 2 (good prognosis). All genes, except one, that wereupregulated in the group with good prognosis were underexpressed in thegroup with poor clinical outcome.

FIG. 11 shows the nearest shrunken centroid of the “43 genesclassifier”: After extending our classifier to 43 genes, a secondupregulated gene appeared in the group with poor prognosis.

DETAILED DESCRIPTION

The invention comprises molecular signatures that function as verysensitive prognostic biomarker for heart failure, heart diseases,myocarditis, and other heart disorders.

Prediction of prognosis remains a major unmet need in new onset heartfailure (HF). While several clinical tests are in use, none accuratelydistinguish between patients with poor vs. excellent survival. Atranscriptomic signature, generated from an endomyocardial biopsy (EMB),serves as a novel prognostic biomarker in HF.

Several aspects of the invention are described below with reference toexample applications for illustration. It should be understood thatnumerous specific details, relationships, and methods are set forth toprovide a full understanding of the invention. One having ordinary skillin the relevant art, however, will readily recognize that the inventioncan be practiced without one or more of the specific details or withother methods. In other instances, well-known structures or operationsare not shown in detail to avoid obscuring the invention. The presentinvention is not limited by the illustrated ordering of acts or events,as some acts may occur in different orders and/or concurrently withother acts or events. Furthermore, not all illustrated acts or eventsare required to implement a methodology in accordance with the presentinvention.

Unless otherwise defined, all terms (including technical and scientificterms) used herein have the same meaning as commonly understood by oneof ordinary skill in the art to which this invention belongs. It will befurther understood that terms, such as those defined in commonly useddictionaries, should be interpreted as having a meaning that isconsistent with their meaning in the context of the relevant art andwill not be interpreted in an idealized or overly formal sense unlessexpressly so defined herein.

All genes, gene names, and gene products disclosed herein are intendedto correspond to homologs from any species for which the compositionsand methods disclosed herein are applicable. Thus, the terms include,but are not limited to genes and gene products from humans and mice. Itis understood that when a gene or gene product from a particular speciesis disclosed, this disclosure is intended to be exemplary only, and isnot to be interpreted as a limitation unless the context in which itappears clearly indicates. Thus, for example, for the genes disclosedherein, which in some embodiments relate to mammalian nucleic acid andamino acid sequences are intended to encompass homologous and/ororthologous genes and gene products from other animals including, butnot limited to other mammals, fish, amphibians, reptiles, and birds. Inpreferred embodiments, the genes or nucleic acid sequences are human.

Definitions

The terminology used herein is for the purpose of describing particularembodiments only and is not intended to be limiting of the invention. Asused herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Furthermore, to the extent that the terms “including”,“includes”, “having”, “has”, “with”, or variants thereof are used ineither the detailed description and/or the claims, such terms areintended to be inclusive in a manner similar to the term “comprising.”

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean within 1 or more than 1 standard deviation,per the practice in the art. Alternatively, “about” can mean a range ofup to 20%, preferably up to 10%, more preferably up to 5%, and morepreferably still up to 1% of a given value. Alternatively, particularlywith respect to biological systems or processes, the term can meanwithin an order of magnitude, preferably within 5-fold, and morepreferably within 2-fold, of a value. Where particular values aredescribed in the application and claims, unless otherwise stated theterm “about” meaning within an acceptable error range for the particularvalue should be assumed.

As used herein, a “molecular signature” or “signature” or “biomarker” or“transcriptomic based biomarker” are used interchangeably herein andrefers to all the biomolecules identified in, for example, Tables 2, and8. Thus, Table 2 comprising the biomolecules listed therein, representsone biomarker or molecular signature; Table 8 comprising thebiomolecules listed therein, represents another one biomarker ormolecular signature; and so forth. As more biomolecules are discovered,each newly identified biomolecules can be assigned to any one or morebiomarker or molecular signature. Each biomolecule can also be removed,reassigned or reallocated to a molecular signature. Thus, in someembodiments the molecular signature comprises at least ten biomolecules.Any one of the biomarkers or combinations thereof can be used in theprognosis of cardiovascular diseases.

The terms “biomolecule” or “markers” are used interchangeably herein andrefer to DNA, RNA (including mRNA, rRNA, tRNA and tmRNA), nucleotides,nucleosides, analogs, polynucleotides, peptides and any combinationsthereof.

A base “position” as used herein refers to the location of a given baseor nucleotide residue within a nucleic acid.

As used herein, the term “array” refers to an ordered spatialarrangement, particularly an arrangement of immobilized biomolecules.

As used herein, the term “addressable array” refers to an array whereinthe individual elements have precisely defined x and y coordinates, sothat a given element at a particular position in the array can beidentified.

As used herein, the terms “probe” and “biomolecular probe” refer to abiomolecule used to detect a complementary biomolecule. Examples includeantigens that detect antibodies, oligonucleotides that detectcomplimentary oligonucleotides, and ligands that detect receptors. Suchprobes are preferably immobilized on a microelectrode comprising asubstrate.

As used herein, the terms “bioarray,” “biochip” and “biochip array”refer to an ordered spatial arrangement of immobilized biomolecules on amicroelectrode arrayed on a solid supporting substrate. Preferred probemolecules include aptamers, nucleic acids, oligonucleotides, peptides,ligands, antibodies and antigens; peptides and proteins are the mostpreferred probe species. Biochips, as used in the art, encompasssubstrates containing arrays or microarrays, preferably ordered arraysand most preferably ordered, addressable arrays, of biological moleculesthat comprise one member of a biological binding pair. Typically, sucharrays are oligonucleotide arrays comprising a nucleotide sequence thatis complementary to at least one sequence that may be or is expected tobe present in a biological sample. Alternatively, and preferably,proteins, peptides or other small molecules can be arrayed in suchbiochips for performing, inter alia, immunological analyses (wherein thearrayed molecules are antigens) or assaying biological receptors(wherein the arrayed molecules are ligands, agonists or antagonists ofsaid receptors).

Expression/amount of a gene, biomolecule, or biomarker in a first sampleis at a level “greater than” the level in a second sample if theexpression level/amount of the gene or biomarker in the first sample isat least about 1 time, 1.2 times, 1.5 times, 1.75 times, 2 times, 3times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times,20 times, 30 times, the expression level/amount of the gene or biomarkerin the second sample or a normal sample. Expression levels/amounts canbe determined based on any suitable criterion known in the art,including but not limited to mRNA, cDNA, proteins, protein fragmentsand/or gene copy. Expression levels/amounts can be determinedqualitatively and/or quantitatively.

By the term “modulate,” it is meant that any of the mentionedactivities, are, e.g., increased, enhanced, increased, agonized (acts asan agonist), promoted, decreased, reduced, suppressed blocked, orantagonized (acts as an antagonist). Modulation can increase activitymore than 1-fold, 2-fold, 3-fold, 5-fold, 10-fold, 100-fold, etc., overbaseline values. Modulation can also decrease its activity belowbaseline values.

The term, “complementary” means that two sequences are complementarywhen the sequence of one can bind to the sequence of the other in ananti-parallel sense wherein the 3′-end of each sequence binds to the5′-end of the other sequence and each A, T(U), G, and C of one sequenceis then aligned with a T(U), A, C, and G, respectively, of the othersequence. Normally, the complementary sequence of the oligonucleotidehas at least 80% or 90%, preferably 95%, most preferably 100%,complementarity to a defined sequence. Preferably, alleles or variantsthereof can be identified. A BLAST program also can be employed toassess such sequence identity.

The term “complementary sequence” as it refers to a polynucleotidesequence, relates to the base sequence in another nucleic acid moleculeby the base-pairing rules. More particularly, the term or like termrefers to the hybridization or base pairing between nucleotides ornucleic acids, such-as, for instance, between the two strands of adouble stranded DNA molecule or between an oligonucleotide primer and aprimer binding site on a single stranded nucleic acid to be sequenced oramplified. Complementary nucleotides are, generally, A and T (or A andU), or C and G. Two single stranded RNA or DNA molecules are said to besubstantially complementary when the nucleotides of one strand,optimally aligned and compared and with appropriate nucleotideinsertions or deletions, pair with at least about 95% of the nucleotidesof the other strand, usually at least about 98%, and more preferablyfrom about 99% to about 100%. Complementary polynucleotide sequences canbe identified by a variety of approaches including use of well-knowncomputer algorithms and software, for example the BLAST program.

An “allele” or “variant” is an alternative form of a gene. Variants mayresult from at least one mutation in the nucleic acid sequence and mayresult in altered mRNAs or in polypeptides whose structure or functionmay or may not be altered. Any given natural or recombinant gene mayhave none, one, or many allelic forms. Common mutational changes thatgive rise to variants are generally ascribed to natural deletions,additions, or substitutions of nucleotides. Each of these types ofchanges may occur alone, or in combination with the others, one or moretimes in a given sequence. The term “variant,” when used in the contextof a polynucleotide sequence, may encompass a polynucleotide sequencerelated to a wild type gene. This definition may also include, forexample, “allelic,” “splice,” “species,” or “polymorphic” variants. Asplice variant may have significant identity to a reference molecule,but will generally have a greater or lesser number of polynucleotidesdue to alternate splicing of exons during mRNA processing. Thecorresponding polypeptide may possess additional functional domains oran absence of domains. Species variants are polynucleotide sequencesthat vary from one species to another. Of particular utility in theinvention are variants of wild type gene products. Variants may resultfrom at least one mutation in the nucleic acid sequence and may resultin altered mRNAs or in polypeptides whose structure or function may ormay not be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes that give rise tovariants are generally ascribed to natural deletions, additions, orsubstitutions of nucleotides. Each of these types of changes may occuralone, or in combination with the others, one or more times in a givensequence.

The resulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species. Polymorphic variants also may encompass “singlenucleotide polymorphisms” (SNPs) or single base mutations in which thepolynucleotide sequence varies by one base. The presence of SNPs may beindicative of, for example, a certain population with a propensity for adisease state, that is susceptibility versus resistance.

Derivative polynucleotides include nucleic acids subjected to chemicalmodification, for example, replacement of hydrogen by an alkyl, acyl, oramino group. Derivatives, e.g., derivative oligonucleotides, maycomprise non-naturally-occurring portions, such as altered sugarmoieties or inter-sugar linkages. Exemplary among these arephosphorothioate and other sulfur containing species which are known inthe art. Derivative nucleic acids may also contain labels, includingradionucleotides, enzymes, fluorescent agents, chemiluminescent agents,chromogenic agents, substrates, cofactors, inhibitors, magneticparticles, and the like.

A “derivative” polypeptide or peptide is one that is modified, forexample, by glycosylation, pegylation, phosphorylation, sulfation,reduction/alkylation, acylation, chemical coupling, or mild formalintreatment. A derivative may also be modified to contain a detectablelabel, either directly or indirectly, including, but not limited to, aradioisotope, fluorescent, and enzyme label.

As used herein, the term “aptamer” or “selected nucleic acid bindingspecies” shall include non-modified or chemically modified RNA or DNA.The method of selection may be by, but is not limited to, affinitychromatography and the method of amplification by reverse transcription(RT) or polymerase chain reaction (PCR).

As used herein, the term “signaling aptamer” shall include aptamers withreporter molecules, preferably a fluorescent dye, appended to anucleotide in such a way that upon conformational changes resulting fromthe aptamer's interaction with a ligand, the reporter molecules yields adifferential signal, preferably a change in fluorescence intensity.

As used herein, the term “fragment or segment”, as applied to a nucleicacid sequence, gene or polypeptide, will ordinarily be at least about 5contiguous nucleic acid bases (for nucleic acid sequence or gene) oramino acids (for polypeptides), typically at least about 10 contiguousnucleic acid bases or amino acids, more typically at least about 20contiguous nucleic acid bases or amino acids, usually at least about 30contiguous nucleic-acid bases or amino acids, preferably at least about40 contiguous nucleic acid bases or amino acids, more preferably atleast about 50 contiguous nucleic acid bases or amino acids, and evenmore preferably at least about 60 to 80 or more contiguous nucleic acidbases or amino acids in length. “Overlapping fragments” as used herein,refer to contiguous nucleic acid or peptide fragments which begin at theamino terminal end of a nucleic acid or protein and end at the carboxyterminal end of the nucleic acid or protein. Each nucleic acid orpeptide fragment has at least about one contiguous nucleic acid or aminoacid position in common with the next nucleic acid or peptide fragment,more preferably at least about three contiguous nucleic acid bases oramino acid positions in common, most preferably at least about tencontiguous nucleic acid bases amino acid positions in common.

“Biological samples” include solid and body fluid samples. Preferably,the sample is obtained from heart. However, the biological samples usedin the present invention can include cells, protein or membrane extractsof cells, blood or biological fluids such as ascites fluid or brainfluid (e.g., cerebrospinal fluid). Examples of solid biological samplesinclude, but are not limited to, samples taken from tissues of thecentral nervous system, bone, breast, kidney, cervix, endometrium,head/neck, gallbladder, parotid gland, prostate, pituitary gland,muscle, esophagus, stomach, small intestine, colon, liver, spleen,pancreas, thyroid, heart, lung, bladder, adipose, lymph node, uterus,ovary, adrenal gland, testes, tonsils and thymus. Examples of “bodyfluid samples” include, but are not limited to blood, serum, semen,prostate fluid, seminal fluid, urine, saliva, sputum, mucus, bonemarrow, lymph, and tears.

“Sample” is used herein in its broadest sense. A sample comprisingpolynucleotides, polypeptides, peptides, antibodies and the like maycomprise a bodily fluid; a soluble fraction of a cell preparation, ormedia in which cells were grown; a chromosome, an organelle, or membraneisolated or extracted from a cell; genomic DNA, RNA, or cDNA,polypeptides, or peptides in solution or bound to a substrate; a cell; atissue; a tissue print; a fingerprint, skin or hair, and the like.

The term “diagnosis”, as used in this specification refers to predictthe type of disease or condition from a set of marker values and/orpatient symptoms. This is in contrast to disease prediction, which is topredict the occurrence of disease before it occurs, and the term“prognosis”, which is to predict disease progression at a future pointin time from one or more indicator value(s) at a previous point in time.

The term “correlating,” as used in this specification refers to aprocess in which a set of examples of clinical inputs from subjects,such as marker levels, and their corresponding outputs, such as whethera subject suffered from heart failure, are related to each other. Thisrelationship can be determined by comparing such examples to examplesfrom a control and/or disease-free population at a later point in time,and selecting those indicators which can differentiate between the twodisease states as a function of time alone or in combination at acertain probability level. The selection process is described herein.The selected markers, each at a certain level range which might be asimple threshold, are said to be correlative or associative with one ofthe disease states. Said correlated markers can be then be used fordisease detection, diagnosis, prognosis and/or treatment outcome.Preferred methods of correlating markers is by performing markerselection as described in detail in the examples section which follows.Methods can include a feature selection algorithm, statistics andclassification by mapping functions described herein. A preferredprobability level is a 3% chance, 5% chance, a 7% chance, a 10% chance,a 15% chance, a 20% chance, a 25% chance, a 30% chance, a 35% chance, a40% chance, a 45% chance, a 50% chance, a 55% chance, a 60% chance, a65% chance, a 70% chance, a 75% chance, a 80% chance, a 85% chance, a90% chance, a 95% chance, and a 100% chance. Each of these values ofprobability is plus or minus 2% or less.

The terms “detecting”, “detect”, “identifying”, “quantifying” includesassaying, quantitating, imaging or otherwise establishing the presenceor absence of the transcriptomic biomarker, or combinations ofbiomolecules comprising the biomarker, and the like, or assaying for,imaging, ascertaining, establishing, or otherwise determining theprognosis and/or diagnosis of heart failure or any other cardiovasculardiseases or conditions.

Transcriptomic Biomarker/Molecular Signatures

In a preferred embodiment, a biomarker for the prognosis of the outcomeof heart failure comprises: biomolecules/nucleic acid sequencescomprising gene sequences: 232669_at (Hypoxia inducible factor 3, alphasubunit), 214951_at (solute carrier family 26, member 10), 243482_at(Epidermal growth factor receptor pathway substrate 15-like 1),226210_s_at (maternally expressed 3), 232159_at (Epidermal growth factorreceptor pathway substrate 15-like 1), 233026_s_at (PDZ domaincontaining), 211996_s_at (KIAA0220-like protein hypothetical gene LOC283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof.

In another preferred embodiment, the detection in a cell or patient of abiomarker (TBB) comprises: nucleic acid sequences/biomoleculescomprising: 232669_at (Hypoxia inducible factor 3, alpha subunit),214951_at (solute carrier family 26, member 10), 243482_at (Epidermalgrowth factor receptor pathway substrate 15-like 1), 226210_s_at(maternally expressed 3), 232159_at (Epidermal growth factor receptorpathway substrate 15-like 1), 233026_s_at (PDZ domain containing),211996_s_at (KIAA0220-like protein hypothetical gene LOC 283846),243774_at (mucin 20, cell surface associated), 242551_at (Chromosome 18open reading frame), 244548_at (Rho GTPase activating protein 26),244208_at (Checkpoint suppressor 1), 239984_at (Sodium channel,voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis, cloneADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof, isdiagnostic of myocardial disorders. Preferably, the nucleic acidsequences, complementary sequences, fragments, alleles, variants,derivatives and/or gene products thereof, are modulated and/orover-expressed at levels by at least between about 1%, 1.2%, 2% 5%, 10%,20% in a cell or patient as compared to levels in a normal cell ornormal subject; more preferably, the nucleic acid sequences,complementary sequences, fragments, alleles, variants, derivativesand/or gene products thereof, are modulated and/or over-expressed byabout 50% in a cell or a patient as compared to levels in a normal cellor normal subject; more preferably, the nucleic acid sequences,complementary sequences, fragments, alleles, variants, derivativesand/or gene products thereof, are modulated and/or over-expressed byabout 75% in a cell or a patient as compared to levels in a normal cellor normal subject.

In another preferred embodiment, a biomarker comprises nucleic acidsequences: 1558458_at (Hypothetical LOC401320), 1560049_at (CUG tripletrepeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNA binding motifprotein 5, RBM5), 201655_s_at (heparan sulfate proteoglycan 2(perlecan), HSPG2), 202379_s_at (natural killer-tumor recognitionsequence, NKTR), 202808_at, 203071_at (sema domain, immunoglobulindomain (Ig), short basic domain, secreted, (semaphorin) 3B, SEMA3B),203748_x_at (RNA binding motif, single stranded interacting protein 1,RBMS1), 203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4,ABCD4), 204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7,MYH6///MYH7), 204978_at (splicing factor, arginine/serine-rich 16,SFRS16), 206209_s_at (carbonic anhydrase IV, CA4), 207541_s_at (exosomecomponent 10, EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at(cysteine-rich protein 2, CRIP2), 209354_at (tumor necrosis factorreceptor superfamily, member 14 TNFRSF14), 210628_x_at (latenttransforming growth factor beta binding protein 4, LTBP4), 211909_x_at(prostaglandin E receptor 3 (subtype EP3), PTGER3), 211996_at(KIAA0220-like protein, nuclear pore complex (LOC23117), 212487_at (Gpatch domain containing 8, GPATCH8), 213946_s_at (obscurin-like 1,OBSL1), 214951_at (solute carrier family 26, member 10, SLC26A10),220219_s_at (leucine rich repeat containing 37A, LRRC37A), 221071_at,221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27),221806_s_at (SET domain containing 5, SETD5), 221833_at (Lon peptidase2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S. cerevisiae), LUC7L),224260_at (CDNA clone IMAGE:4478733), 225562_at (AS p21 proteinactivator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156 (from cloneDKFZp762N156), 227968_at (Parkinson disease 7 domain containing 1,PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9, MRPS9),229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis, cloneADKA03430), 238185_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 241597_at (Arginine-glutamic acid dipeptide (RE)repeats, RERE), 242551_at (Chromosome 18 open reading frame 1, C18orf1),244208_at (Checkpoint suppressor 1, CHES1), 244494_at (Zinc finger,DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPase activatingprotein 26, ARHGAP26) complementary sequences, fragments, alleles,variants, derivatives and/or gene products thereof.

In another preferred embodiment, a biochip comprises a molecularsignature/biomarker comprising nucleic acid sequences: a biomarkercomprises gene sequences: 232669_at (Hypoxia inducible factor 3, alphasubunit), 214951_at (solute carrier family 26, member 10), 243482_at(Epidermal growth factor receptor pathway-substrate 15-like 1),226210_s_at (maternally expressed 3), 232159_at (Epidermal growth factorreceptor pathway substrate 15-like 1), 233026_s_at (PDZ domaincontaining), 211996_s_at (KIAA0220-like protein hypothetical gene LOC283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof.

In another preferred embodiment a biochip comprises nucleic acidsequences: 1558458_at (Hypothetical LOC401320), 1560049_at (CUG tripletrepeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNA binding motifprotein 5, RBM5), 201655_s_at (heparan sulfate proteoglycan 2(perlecan), HSPG2), 202379_s_at (natural killer-tumor recognitionsequence, NKTR), 202808_at, 203071_at (sema domain, immunoglobulindomain (Ig), short basic domain, secreted, (semaphorin) 3B, SEMA3B),203748_x_at (RNA binding motif, single stranded interacting protein 1,RBMS1), 203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4,ABCD4), 204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7,MYH6/II MYH7), 204978_at (splicing factor, arginine/serine-rich 16,SFRS16), 206209_s_at (carbonic anhydrase IV, CA4), 207541_s_at (exosomecomponent 10, EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at(cysteine-rich protein 2, CRIP2), 209354_at (tumor necrosis factorreceptor superfamily, member 14 TNFRSF14), 210628_x_at (latenttransforming growth factor beta binding protein 4, LTBP4), 211909_x_at(prostaglandin E receptor 3 (subtype EP3), PTGER3), 211996_at(KIAA0220-like protein, nuclear pore complex (LOC23117), 212487_at (Gpatch domain containing 8, GPATCH8), 213946_s_at (obscurin-like 1,OBSL1), 214951_at (solute carrier family 26, member 10, SLC26A10),220219_s_at (leucine rich repeat containing 37A, LRRC37A), 221071_at,221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27),221806_s_at (SET domain containing 5, SETD5), 221833_at (Lon peptidase2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S. cerevisiae), LUC7L),224260_at (CDNA clone IMAGE:4478733), 225562_at (AS p21 proteinactivator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156 (from cloneDKFZp762N156), 227968_at (Parkinson disease 7 domain containing 1,PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9, MRPS9),229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis, cloneADKA03430), 238185_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 241597_at (Arginine-glutamic acid dipeptide (RE)repeats, RERE), 242551_at (Chromosome 18 open reading frame 1, C18orf1),244208_at (Checkpoint suppressor 1, CHES1), 244494_at (Zinc finger,DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPase activatingprotein 26, ARHGAP26) complementary sequences, fragments, alleles,variants, derivatives and/or gene products thereof.

In another preferred embodiment, a method of prognosis of heart failurecomprises identifying in a biological sample from a patient a molecularsignature comprising a transcriptomic based biomarker (TBB): a biomarkercomprises gene sequences: 232669_at (Hypoxia inducible factor 3, alphasubunit), 214951_at (solute carrier family 26, member 10), 243482_at(Epidermal growth factor receptor pathway substrate 15-like 1),226210_s_at (maternally expressed 3), 232159_at (Epidermal growth factorreceptor pathway substrate 15-like 1), 233026_s_at (PDZ domaincontaining), 211996_s_at (KIAA0220-like protein hypothetical gene LOC283846), 243774_at (mucin 20, cell surface associated), 242554_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof; assessingthe probability of identification of each component gene in each sample;and assigning each to a class.

In another preferred embodiment, a method of predicting the clinicaloutcome and prognosis in heart failure comprises detection of abiomarker comprising: 1558458_at (Hypothetical LOC401320), 1560049_at(CUG triplet repeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNAbinding motif protein 5, RBM5), 201655_s_at (heparan sulfateproteoglycan 2 (perlecan), HSPG2), 202379_s_at (natural killer-tumorrecognition sequence, NKTR), 202808_at, 203071_at (sema domain,immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin)3B, SEMA3B), 203748_x_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 203981_s_at (ATP-binding cassette, sub-family D(ALD), member 4, ABCD4), 204737_s_at (myosin, heavy chain 6, myosin,heavy chain 7, MYH6///MYH7), 204978_at (splicing factor,arginine/serine-rich 16, SFRS16), 206209_s_at (carbonic anhydrase IV,CA4), 207541_s_at (exosome component 10, EXOSC10), 207798_s_at (ataxin2-like, ATXN2L), 208978_at (cysteine-rich protein 2, CRIP2), 209354_at(tumor necrosis factor receptor superfamily, member 14 TNFRSF14),210628_x_at (latent transforming growth factor beta binding protein 4,LTBP4), 211909_x_at (prostaglandin E receptor 3 (subtype EP3), PTGER3),211996_at (KIAA0220-like protein, nuclear pore complex (LOC23117),212487_at (G patch domain containing 8, GPATCH8), 213946_s_at(obscurin-like 1, OBSL1), 214951_at (solute carrier family 26, member10, SLC26A10), 220219_s_at (leucine rich repeat containing 37A,LRRC37A), 221071_at, 221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide27DDX27), 221806_s_at (SET domain containing 5, SETD5), 221833_at (Lonpeptidase 2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S.cerevisiae), LUC7L), 224260_at (CDNA clone IMAGE:4478733), 225562_at (ASp21 protein activator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156(from clone DKFZp762N156), 227968_at (Parkinson disease 7 domaincontaining 1, PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9,MRPS9), 229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis,clone ADKA03430), 238185_at (RNA binding motif, single strandedinteracting protein 1, RBMS1), 241597_at (Arginine-glutamic aciddipeptide (RE) repeats, RERE), 242551_at (Chromosome 18 open readingframe 1, C18orf1), 244208_at (Checkpoint suppressor 1, CHES1), 244494_at(Zinc finger, DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPaseactivating protein 26, ARHGAP26) complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof.

In a preferred embodiment, phenotype specificity is identified bycreating a classifier in a training set comprising about 66% of dataobtained, with subsequent validation in a test set comprising about 33%of data obtained and defining a phenotype specific nearest shrunkencentroid for classification.

In another preferred embodiment, a method of identifying anddistinguishing between patients at a high risk of heart disease andpatients with a good prognosis for recovery comprising: identifying in abiological sample from a patient a molecular signature comprising atranscriptomic based biomarker (TBB): a biomarker comprises genesequences: 232669_at (Hypoxia inducible factor 3, alpha subunit),214951_at (solute carrier family 26, member 10), 243482_at (Epidermalgrowth factor receptor pathway substrate 15-like 1), 226210_s_at(maternally expressed 3), 232159_at (Epidermal growth factor receptorpathway substrate 15-like 1), 233026_s_at (PDZ domain containing),211996_s_at (KIAA0220-like protein hypothetical gene LOC 283846),243774_at (mucin 20, cell surface associated), 242551_at (Chromosome 18open reading frame), 244548_at (Rho GTPase activating protein 26),244208_at (Checkpoint suppressor 1), 239984_at (Sodium channel,voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis, cloneADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof; assessingthe probability of identification of each component gene in each sample;assigning each to a class; and, identifying and distinguishing betweenpatients at a high risk of heart disease and patients with a goodprognosis for recovery.

In another preferred embodiment, a method of identifying anddistinguishing between patients at a high risk of heart disease andpatients with a good prognosis for recovery comprising: identifying in abiological sample from a patient a molecular signature comprising atranscriptomic based biomarker (TBB): a biomarker comprises genesequences: 1558458_at (Hypothetical LOC401320), 1560049_at (CUG tripletrepeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNA binding motifprotein 5, RBM5), 201655_s_at (heparan sulfate proteoglycan 2(perlecan), HSPG2), 202379_s_at (natural killer-tumor recognitionsequence, NKTR), 202808_at, 203071_at (sema domain, immunoglobulindomain (Ig), short basic domain, secreted, (semaphorin) 3B, SEMA3B),203748_x_at (RNA binding motif, single stranded interacting protein 1,RBMS1), 203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4,ABCD4), 204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7,MYH6///MYH7), 204978_at (splicing factor, arginine/serine-rich 16,SFRS16), 206209_s_at (carbonic anhydrase IV, CA4), 207541_s_at (exosomecomponent 10, EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at(cysteine-rich protein 2, CRIP2), 209354_at (tumor necrosis factorreceptor superfamily, member 14 TNFRSF14), 210628_x_at (latenttransforming growth factor beta binding protein 4, LTBP4), 211909_x_at(prostaglandin E receptor 3 (subtype EP3), PTGER3), 211996_at(KIAA0220-like protein, nuclear pore complex (LOC23117), 212487_at (Gpatch domain containing 8, GPATCH8), 213946_s_at (obscurin-like 1,OBSL1), 214951_at (solute carrier family 26, member 10, SLC26A10),220219_s_at (leucine rich repeat containing 37A, LRRC37A), 221071_at,221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27),221806_s_at (SET domain containing 5, SETD5), 221833_at (Lon peptidase2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S. cerevisiae), LUC7L),224260_at (CDNA clone IMAGE:4478733), 225562_at (AS p21 proteinactivator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156 (from cloneDKFZp762N156), 227968_at (Parkinson disease 7 domain containing 1,PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9, MRPS9),229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis, cloneADKA03430), 238185_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 241597_at (Arginine-glutamic acid dipeptide (RE)repeats, RERE), 242551_at (Chromosome 18 open reading frame 1, C18orf1),244208_at (Checkpoint suppressor 1, CHES1), 244494_at (Zinc finger,DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPase activatingprotein 26, ARHGAP26) complementary sequences, fragments, alleles,variants, derivatives and/or gene products thereof; assessing theprobability of identification of each component gene in each sample;assigning each to a class; and, identifying and distinguishing betweenpatients at a high risk of heart disease and patients with a goodprognosis for recovery.

In another preferred embodiment, prediction of prognosis of heartfailure comprises detecting at least ten molecules having gene sequencescomprising: 232669_at (Hypoxia inducible factor 3, alpha subunit),214951_at (solute carrier family 26, member 10), 243482_at (Epidermalgrowth factor receptor pathway substrate 15-like 1), 226210_s_at(maternally expressed 3), 232159_at (Epidermal growth factor receptorpathway substrate 15-like 1), 233026_s_at (PDZ domain containing),211996_s_at (KIAA0220-like protein hypothetical gene LOC 283846),243774_at (mucin 20, cell surface associated), 242551_at (Chromosome 18open reading frame), 244548_at (Rho GTPase activating protein 26),244208_at (Checkpoint suppressor 1), 239984_at (Sodium channel,voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis, cloneADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof.

In another preferred embodiment, prediction of prognosis of heartfailure comprises detecting at least nine molecules having genesequences comprising: 1558458_at (Hypothetical LOC401320), 1560049_at(CUG triplet repeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNAbinding motif protein 5, RBM5), 201655_s_at (heparan sulfateproteoglycan 2 (perlecan), HSPG2), 202379_s_at (natural killer-tumorrecognition sequence, NKTR), 202808_at, 203071_at (sema domain,immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin)3B, SEMA3B), 203748_x_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 203981_s_at (ATP-binding cassette, sub-family D(ALD), member 4, ABCD4), 204737_s_at (myosin, heavy chain 6, myosin,heavy chain 7, MYH6///MYH7), 204978_at (splicing factor,arginine/serine-rich 16, SFRS16), 206209_s_at (carbonic anhydrase IV,CA4), 207541_s_at (exosome component 10, EXOSC10), 207798_s_at (ataxin2-like, ATXN2L), 208978_at (cysteine-rich protein 2, CRIP2), 209354_at(tumor necrosis factor receptor superfamily, member 14 TNFRSF14),210628_x_at (latent transforming growth factor beta binding protein 4,LTBP4), 211909_x_at (prostaglandin E receptor 3 (subtype EP3), PTGER3),211996_at (KIAA0220-like protein, nuclear pore complex (LOC23117),212487_at (G patch domain containing 8, GPATCH8), 213946_s_at(obscurin-like 1, OBSL1), 214951_at (solute carrier family 26, member10, SLC26A10), 220219_s_at (leucine rich repeat containing 37A,LRRC37A), 221071_at, 221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide27DDX27), 221806_s_at (SET domain containing 5, SETD5), 221833_at (Lonpeptidase 2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S.cerevisiae), LUC7L), 224260_at (CDNA clone IMAGE:4478733), 225562_at (ASp21 protein activator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156(from clone DKFZp762N156), 227968_at (Parkinson disease 7 domaincontaining 1, PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9,MRPS9), 229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis,clone ADKA03430), 238185_at (RNA binding motif, single strandedinteracting protein 1, RBMS1), 241597_at (Arginine-glutamic aciddipeptide (RE) repeats, RERE), 242551_at (Chromosome 18 open readingframe 1, C18orf1), 244208_at (Checkpoint suppressor 1, CHES1), 244494_at(Zinc finger, DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPaseactivating protein 26, ARHGAP26) complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof.

In another preferred embodiment, a biochip composition comprising genesequences: a biomarker comprises gene sequences: 232669_at (Hypoxiainducible factor 3, alpha subunit), 214951_at (solute carrier family 26,member 10), 243482_at (Epidermal growth factor receptor pathwaysubstrate 15-like 1), 226210_s_at (maternally expressed 3), 232159_at(Epidermal growth factor receptor pathway substrate 15-like 1),233026_s_at (PDZ domain containing), 211996_s_at (KIAA0220-like proteinhypothetical gene LOC 283846), 243774_at (mucin 20, cell surfaceassociated), 242551_at (Chromosome 18 open reading frame), 244548_at(Rho GTPase activating protein 26), 244208_at (Checkpoint suppressor 1),239984_at (Sodium channel, voltage-gated, type VII, alpha), 230683_at(CDNA:FLJ20892 fis, clone ADKA03430), 214869_at (apolipoprotein L, 6),241597_at (Arginine-glutamic acid dipeptide (RE) repeats), 235887_at(Smg-6 homolog, nonsense mediated mRNA decay factor (C. elegans)),229957_at (transmembrane protein 91), 223546_x_at (LUC7L-like (S.cerevisiae)), 239567_at (Rho GTPase activating protein 10), 242194_at(Cullin 4A), 1558525_at (hypothetical protein LOC283901), 227178_at (CUGtriplet repeat, RNA binding protein 2), 228198_s_at (Mitochondrialribosomal protein S9), 202379_s_at (natural killer-tumor recognitionsequence), 224260_at (CDNA clone), 238643_at (Neuroblastoma, suppressionof tumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)),227968_at (Parkinson disease 7 domain containing 1), 233197_at(kelch-like 9 (Drosophila)), 244512_at (transcribed locus stronglysimilar to XP_0010813421), 233443_at (hypothetical protein LOC389362),231275_at (FLJ42875 protein), 226419_s_at (hypothetical proteinLOC64546), 201221_s_at small nuclear ribonucleoprotein 70 kDapolypeptide), 209354_at (tumor necrosis factor receptor family member14), 226571_s_at (protein tyrosine phosphatase receptor type, S),220728_at (EST), 203071_at (sema domain, immunoglobulin domain (Ig),short basic domain), 213946_s_at obscurin-like 1, similar to titinisoform N2-B), 201394_s_at (RNA binding motif protein 5), 203748_x_at(RNA binding motif, single stranded interacting protein 1), 223147_s_at(WD repeat domain 33), 213773_x_at (NOL/NOP2/Sun domain family, member5), 1560049_at CUG triplet repeat, RNA binding protein 2), 243974_at(CDNA clone IMAGE:4821815), 201510_at E74-like factor 3 (ets domaintranscription factor, epithelial specific), complementary sequences,fragments, alleles, variants, derivatives and/or gene products thereof.

In one embodiment, the biochip comprises at least ten biomoleculesselected from a biomarker comprises gene sequences: 232669_at (Hypoxiainducible factor 3, alpha subunit), 214951_at (solute carrier family 26,member 10), 243482_at (Epidermal growth factor receptor pathwaysubstrate 15-like 1), 226210_s_at (maternally expressed 3), 232159_at(Epidermal growth factor receptor pathway substrate 15-like 1),233026_s_at (PDZ domain containing), 211996_s_at (KIAA0220-like proteinhypothetical geneLOC 283846), 243774_at (mucin 20, cell surfaceassociated), 242551_at (Chromosome 18 open reading frame), 244548_at(Rho GTPase activating protein 26), 244208_at (Checkpoint suppressor 1),239984_at (Sodium channel, voltage-gated, type VII, alpha), 230683_at(CDNA:FLJ20892 fis, clone ADKA03430), 214869_at (apolipoprotein L, 6),241597_at (Arginine-glutamic acid dipeptide (RE) repeats), 235887_at(Smg-6 homolog, nonsense mediated mRNA decay factor (C. elegans)),229957_at (transmembrane protein 91), 223546_x_at (LUC7L-like (S.cerevisiae)), 239567_at (Rho GTPase activating protein 10), 242194_at(Cullin 4A), 1558525_at (hypothetical protein LOC283901), 227178_at (CUGtriplet repeat, RNA binding protein 2), 228198_s_at (Mitochondrialribosomal protein S9), 202379_s_at (natural killer-tumor recognitionsequence), 224260_at (CDNA clone), 238643_at (Neuroblastoma, suppressionof tumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)),227968_at (Parkinson disease 7 domain containing 1), 233197_at(kelch-like 9 (Drosophila)), 244512_at (transcribed locus stronglysimilar to XP_0010813421), 233443_at (hypothetical protein LOC389362),231275_at (FLJ42875 protein), 226419_s_at (hypothetical proteinLOC64546), 201221_s_at small nuclear ribonucleoprotein 70 kDapolypeptide), 209354_at (tumor necrosis factor receptor family member14), 226571_s_at (protein tyrosine phosphatase receptor type, S),220728_at (EST), 203071_at (sema domain, immunoglobulin domain (Ig),short basic domain), 213946_s_at obscurin-like 1, similar to titinisoform N2-B), 201394_s_at (RNA binding motif protein 5), 203748_x_at(RNA binding motif, single stranded interacting protein 1), 223147_s_at(WD repeat domain 33), 213773_x_at (NOL/NOP2/Sun domain family, member5), 1560049_at CUG triplet repeat, RNA binding protein 2), 243974_at(CDNA clone IMAGE:4821815), 201510_at E74-like factor 3 (ets domaintranscription factor, epithelial specific), complementary sequences,fragments, alleles, variants, derivatives and/or gene products thereof.

In another preferred embodiment, a biochip composition comprising genesequences: a biomarker comprises gene sequences: 1558458_at(Hypothetical LOC401320), 1560049_at (CUG triplet repeat, RNA bindingprotein 2, CUGBP2), 201394_s_at (RNA binding motif protein 5, RBM5),201655_s_at (heparan sulfate proteoglycan 2 (perlecan), HSPG2),202379_s_at (natural killer-tumor recognition sequence, NKTR),202808_at, 203071_at (sema domain, immunoglobulin domain (Ig), shortbasic domain, secreted, (semaphorin) 3B, SEMA3B), 203748_x_at (RNAbinding motif, single stranded interacting protein 1, RBMS1),203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4, ABCD4),204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7, MYH6///MYH7),204978_at (splicing factor, arginine/serine-rich 16, SFRS6), 206209_s_at(carbonic anhydrase IV, CA4), 207541_s_at (exosome component 10,EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at (cysteine-richprotein 2, CRIP2), 209354_at (tumor necrosis factor receptorsuperfamily, member 14 TNFRSF14), 210628_x_at (latent transforminggrowth factor beta binding protein 4, LTBP4), 211909_x_at (prostaglandinE receptor 3 (subtype EP3), PTGER3), 211996_at (KIAA0220-like protein,nuclear pore complex (LOC23117), 212487_at (G patch domain containing 8,GPATCH8), 213946_s_at (obscurin-like 1, OBSL1), 214951_at (solutecarrier family 26, member 10, SLC26A10), 220219_s_at (leucine richrepeat containing 37A, LRRC37A), 221071_at, 221780_s_at (DEAD(Asp-Glu-Ala-Asp) box polypeptide 27DDX27), 221806_s_at (SET domaincontaining 5, SETD5), 221833_at (Lon peptidase 2, peroxisomal, LONP2),223546_x_at (LUC7-like (S. cerevisiae), LUC7L), 224260_at (CDNA cloneIMAGE:4478733), 225562_at (AS p21 protein activator 3, RASA3), 226040_at(MRNA; cDNA DKFZp762N156 (from clone DKFZp762N156), 227968_at (Parkinsondisease 7 domain containing 1, PDDC1), 228198_s_at (Mitochondrialribosomal protein S9, MRPS9), 229830_at (Transcribed locus), 230683_at(CDNA: FLJ20892 fis, clone ADKA03430), 238185_at (RNA binding motif,single stranded interacting protein 1, RBMS1), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats, RERE), 242551_at(Chromosome 18 open reading frame 1, C18orf1), 244208_at (Checkpointsuppressor 1, CHES1), 244494_at (Zinc finger, DHHC-type containing 1,ZDHHC1), and 244548_at (Rho GTPase activating protein 26, ARHGAP26)complementary sequences, fragments, alleles, variants, derivativesand/or gene products thereof.

In a preferred embodiment, a biochip comprises at least nine moleculescomprising: 1558458_at (Hypothetical LOC401320), 1560049_at (CUG tripletrepeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNA binding motifprotein 5, RBM5), 201655_s_at (heparan sulfate proteoglycan 2(perlecan), HSPG2), 202379_s_at (natural killer-tumor recognitionsequence, NKTR), 202808_at, 203071_at (sema domain, immunoglobulindomain (Ig), short basic domain, secreted, (semaphorin) 3B, SEMA3B),203748_x_at (RNA binding motif, single stranded interacting protein 1,RBMS1), 203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4,ABCD4), 204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7,MYH6/II MYH7), 204978_at (splicing factor, arginine/serine-rich 16,SFRS16), 206209_s_at (carbonic anhydrase IV, CA4), 207541_s_at (exosomecomponent 10, EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at(cysteine-rich protein 2, CRIP2), 209354_at (tumor necrosis factorreceptor superfamily, member 14 TNFRSF4), 210628_x_at (latenttransforming growth factor beta binding protein 4, LTBP4), 211909_x_at(prostaglandin E receptor 3 (subtype EP3), PTGER3), 211996_at(KIAA0220-like protein, nuclear pore complex (LOC23117), 212487_at (Gpatch domain containing 8, GPATCH8), 213946_s_at (obscurin-like 1,OBSL1), 214951_at (solute carrier family 26, member 10, SLC26A10),220219_s_at (leucine rich repeat containing 37A, LRRC37A), 221071_at,221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27),221806_s_at (SET domain containing 5, SETD5), 221833_at (Lon peptidase2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S. cerevisiae), LUC7L),224260_at (CDNA clone IMAGE:4478733), 225562_at (AS p21 proteinactivator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156 (from cloneDKFZp762N156), 227968_at (Parkinson disease 7 domain containing 1,PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9, MRPS9),229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis, cloneADKA03430), 238185_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 241597_at (Arginine-glutamic acid dipeptide (RE)repeats, RERE), 242551_at (Chromosome 18 open reading frame 1, C18orf1),244208_at (Checkpoint suppressor 1, CHES1), 244494_at (Zinc finger,DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPase activatingprotein 26, ARHGAP26) complementary sequences, fragments, alleles,variants, derivatives and/or gene products thereof.

In another preferred embodiment, a method of predicting a prognosis ofnew onset heart failure, heart disease or conditions thereof comprising:identifying in a biological sample from a patient a biomarker comprisinggene sequences: a biomarker comprises gene sequences: 232669_at (Hypoxiainducible factor 3, alpha subunit), 214951_at (solute carrier family 26,member 10), 243482_at (Epidermal growth factor receptor pathwaysubstrate 15-like 1), 226210_s_at (maternally expressed 3), 232159_at(Epidermal growth factor receptor pathway substrate 15-like 1),233026_s_at (PDZ domain containing), 211996_s_at (KIAA0220-like proteinhypothetical gene LOC 283846), 243774_at (mucin 20, cell surfaceassociated), 242551_at (Chromosome 18 open reading frame), 244548_at(Rho GTPase activating protein 26), 244208_at (Checkpoint suppressor 1),239984_at (Sodium channel, voltage-gated, type VII, alpha), 230683_at(CDNA:FLJ20892 fis, clone ADKA03430), 214869_at (apolipoprotein L, 6),241597_at (Arginine-glutamic acid dipeptide (RE) repeats), 235887_at(Smg-6 homolog, nonsense mediated mRNA decay factor (C. elegans)229957_at (transmembrane protein 91), 223546_x_at (LUC7L-like (S.cerevisiae)), 239567_at (Rho GTPase activating protein 10), 242194_at(Cullin 4A), 1558525_at (hypothetical protein LOC283901), 227178_at (CUGtriplet repeat, RNA binding protein 2), 228198_s_at (Mitochondrialribosomal protein S9), 202379_s_at (natural killer-tumor recognitionsequence), 224260_at (CDNA clone), 238643_at (Neuroblastoma, suppressionof tumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)),227968_at (Parkinson disease 7 domain containing 1), 233197_at(kelch-like 9 (Drosophila)), 244512_at (transcribed locus stronglysimilar to XP_0010813421), 233443_at (hypothetical protein LOC389362),231275_at (FLJ42875 protein), 226419_s_at (hypothetical proteinLOC64546), 201221_s_at small nuclear ribonucleoprotein 70 kDapolypeptide), 209354_at (tumor necrosis factor receptor family member14), 226571_s_at (protein tyrosine phosphatase receptor type, S),220728_at (EST), 203071_at (sema domain, immunoglobulin domain (Ig),short basic domain), 213946_s_at obscurin-like 1, similar to titinisoform N2-B), 201394_s_at (RNA binding motif protein 5), 203748_x_at(RNA binding motif, single stranded interacting protein 1), 223147_s_at(WD repeat domain 33), 213773_x_at (NOL/NOP2/Sun domain family, member5), 1560049_at CUG triplet repeat, RNA binding protein 2), 243974_at(CDNA clone IMAGE:4821815), 201510_at E74-like factor 3 (ets domaintranscription factor, epithelial specific), complementary sequences,fragments, alleles, variants, derivatives and/or gene products thereof;and, assessing the probability of identification of each component genein each sample; and assigning each to a class. An example of a preferredmethod is detailed in the examples which follow.

In another preferred embodiment, a method of identifying anddistinguishing between patients at a high risk of heart disease andpatients with a good prognosis for recovery comprising: identifying abiomarker comprising gene sequences comprising: a biomarker comprisesgene sequences: 232669_at (Hypoxia inducible factor 3, alpha subunit),214951_at (solute carrier family 26, member 10), 243482_at (Epidermalgrowth factor receptor pathway substrate 15-like 1), 226210_s_at(maternally expressed 3), 232159_at (Epidermal growth factor receptorpathway substrate 15-like 1), 233026_s_at (PDZ domain containing),211996_s_at (KIAA0220-like protein hypothetical gene LOC 283846),243774_at (mucin 20, cell surface associated), 242551_at (Chromosome 18open reading frame), 244548_at (Rho GTPase activating protein 26),244208_at (Checkpoint suppressor 1), 239984_at (Sodium channel,voltage-gated, type VII, alpha), 230683_at CDNA:FLJ20892 fis, cloneADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof; assessingthe probability of identification of each component gene in each sample;assigning each to a class; and, identifying and distinguishing betweenpatients at a high risk of heart disease and patients with a goodprognosis for recovery.

In another preferred embodiment, a method of identifying anddistinguishing between patients at a high risk of heart disease andpatients with a good prognosis for recovery comprising: identifying abiomarker comprising gene sequences comprising: a biomarker comprisesgene sequences: 1558458_at (Hypothetical LOC401320), 1560049_at (CUGtriplet repeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNA bindingmotif protein 5, RBM5), 201655_s_at (heparan sulfate proteoglycan 2(perlecan), HSPG2), 202379_s_at (natural killer-tumor recognitionsequence, NKTR), 202808_at, 203071_at (sema domain, immunoglobulindomain (Ig), short basic domain, secreted, (semaphorin) 3B, SEMA3B),203748_x_at (RNA binding motif, single stranded interacting protein 1,RBMS1), 203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4,ABCD4), 204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7,MYH6///MYH7), 204978_at (splicing factor, arginine/serine-rich 16,SFRS16), 206209_s_at (carbonic anhydrase IV, CA4), 207541_s_at (exosomecomponent 10, EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at(cysteine-rich protein 2, CRIP2), 209354_at (tumor necrosis factorreceptor superfamily, member 14 TNFRSF14), 210628_x_at (latenttransforming growth factor beta binding protein 4, LTBP4), 211909_x_at(prostaglandin E receptor 3 (subtype EP3), PTGER3), 211996_at(KIAA0220-like protein, nuclear pore complex (LOC23117), 212487_at (Gpatch domain containing 8, GPATCH8), 213946_s_at (obscurin-like 1,OBSL1), 214951_at (solute carrier family 26, member 10, SLC26A10),220219_s_at (leucine rich repeat containing 37A, LRRC37A), 221071_at,221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27),221806_s_at (SET domain containing 5, SETD5), 221833_at (Lon peptidase2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S. cerevisiae), LUC7L),224260_at (CDNA clone IMAGE:4478733), 225562_at (AS p21 proteinactivator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156 (from cloneDKFZp762N156), 227968_at (Parkinson disease 7 domain containing 1,PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9, MRPS9),229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis, cloneADKA03430), 238185_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 241597_at (Arginine-glutamic acid dipeptide (RE)repeats, RERE), 24255.1_at (Chromosome 18 open reading frame 1,C18orf1), 244208_at (Checkpoint suppressor 1, CHES1), 244494_at (Zincfinger, DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPaseactivating protein 26, ARHGAP26) complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof.

Alternative Methods and Materials for Identifying Molecular Signaturesor Transcriptomic Biomarkers

Detection of Nucleic Acids and Proteins as Markers:

In preferred embodiments, each biomarker is detected on chip basedmethods such as those described in detail in the examples which follow.In order to provide accurate prognosis and diagnosis of heart failurepatients with good prognostic outcome, cardiac disorders and diseases,for example, heart failure, myocarditis, idiopathic cardiomyopathy andthe like. Other methods are also known in the art and one or moremethods can be utilized.

The methods and assays disclosed herein are directed to the examinationof expression of transcriptomic biomarkers in a mammalian tissue or cellsample, wherein the determination of that expression of one or more suchtranscriptomic biomarkers is predictive of prognostic outcome ordiagnostic of cardiac and cardiovascular diseases and disorders, such asfor example, myocarditis, Coronary Heart Disease, angina, Acute CoronarySyndrome, Aortic Aneurysm and Dissection, arrhythmias, Cardiomyopathy,Congenital Heart Disease, congestive heart failure or chronic heartfailure, pericarditis, and the like. The Molecular signatures orTranscriptomic biomarker comprise the biomolecules identified in Table2. As more biomolecules are discovered, each newly identifiedbiomolecules can be assigned to any one or more biomarker or molecularsignature. Each biomolecule can also be removed, reassigned orreallocated to a molecular signature.

Preferred embodiments in the identification of biomolecules, analyticalmethods etc, are described in detail in the Examples which follow.

Microarrays:

In general, using nucleic acid microarrays, test and control mRNAsamples from test and control tissue samples are reverse transcribed andlabeled to generate cDNA probes. The probes are then hybridized to anarray of nucleic acids immobilized on a solid support. The array isconfigured such that the sequence and position of each member of thearray is known. For example, a selection of genes that have potential tobe expressed in certain disease states may be arrayed on a solidsupport. Hybridization of a labeled probe with a particular array memberindicates that the sample from which the probe was derived expressesthat gene. Differential gene expression analysis of disease tissue canprovide valuable information. Microarray technology utilizes nucleicacid hybridization techniques and computing technology to evaluate themRNA expression profile of thousands of genes within a singleexperiment. (see, e.g., WO 01/75166 published Oct. 11, 2001; (See, forexample, U.S. Pat. Nos. 5,700,637, 5,445,934, and 5,807,522, Lockart,Nature Biotechnology, 14:1675-1680 (1996); Cheung, V. G. et al., NatureGenetics 21(Suppl):15-19 (1999) for a discussion of array fabrication).DNA microarrays are miniature arrays containing gene fragments that areeither synthesized directly onto or spotted onto glass or othersubstrates. Thousands of genes are usually represented in a singlearray. A typical microarray experiment involves the following steps: 1)preparation of fluorescently labeled target from RNA isolated from thesample, 2) hybridization of the labeled target to the microarray, 3)washing, staining, and scanning of the array, 4) analysis of the scannedimage and 5) generation of gene expression profiles. Currently two maintypes of DNA microarrays are being used: oligonucleotide (usually 25 to70 mers) arrays and gene expression arrays containing PCR productsprepared from cDNAs. In forming an array, oligonucleotides can be eitherprefabricated and spotted to the surface or directly synthesized on tothe surface (in situ). The Affymetrix GeneChip™ system is a commerciallyavailable microarray system which comprises arrays fabricated by directsynthesis of oligonucleotides on a glass surface.

Probe/Gene Arrays:

Oligonucleotides, usually 25 mers, are directly synthesized onto a glasswafer by a combination of semiconductor-based photolithography and solidphase chemical synthesis technologies. Each array contains up to 400,000different oligonucleotides and each oligonucleotide is present inmillions of copies. Since oligonucleotide probes are synthesized inknown locations on the array, the hybridization patterns and signalintensities can be interpreted in terms of gene identity and relativeexpression levels by the Affymetrix Microarray Suite software. Each geneis represented on the array by a series of different oligonucleotideprobes. Each probe pair consists of a perfect match oligonucleotide anda mismatch oligonucleotide. The perfect match probe has a sequenceexactly complimentary to the particular gene and thus measures theexpression of the gene. The mismatch probe differs from the perfectmatch probe by a single base substitution at the center base position,disturbing the binding of the target gene transcript. This helps todetermine the background and nonspecific hybridization that contributesto the signal measured for the perfect match oligonucleotide. TheMicroarray Suite software subtracts the hybridization intensities of themismatch probes from those of the perfect match probes to determine theabsolute or specific intensity value for each probe set. Probes arechosen based on current information from GenBank and other nucleotiderepositories. The sequences are believed to recognize unique regions ofthe 3′ end of the gene. A GeneChip Hybridization Oven (“rotisserie”oven) is used to carry out the hybridization of up to 64 arrays at onetime. The fluidics station performs washing and staining of the probearrays. It is completely automated and contains four modules, with eachmodule holding one probe array. Each module is controlled independentlythrough Microarray Suite software using preprogrammed fluidicsprotocols. The scanner is a confocal laser fluorescence scanner whichmeasures fluorescence intensity emitted by the labeled cRNA bound to theprobe arrays. The computer workstation with Microarray Suite softwarecontrols the fluidics station and the scanner. Microarray Suite softwarecan control up to eight fluidics stations using preprogrammedhybridization, wash, and stain protocols for the probe array. Thesoftware also acquires and converts hybridization intensity data into apresence/absence call for each gene using appropriate algorithms.Finally, the software detects changes in gene expression betweenexperiments by comparison analysis and formats the output into .txtfiles, which can be used with other software programs for further dataanalysis.

The expression of a selected biomarker may also be assessed by examininggene deletion or gene amplification. Gene deletion or amplification maybe measured by any one of a wide variety of protocols known in the art,for example, by conventional Southern blotting, Northern blotting toquantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Sci.USA, 77:5201-5205 (1980)), dot blotting (DNA analysis), or in situhybridization (e.g., FISH), using an appropriately labeled probe,cytogenetic methods or comparative genomic hybridization (CGH) using anappropriately labeled probe.

Detection of Polypeptides:

In another embodiment of the present invention, a polypeptidecorresponding to a marker is detected. A preferred agent for detecting apolypeptide of the invention is an antibody or aptamer capable ofbinding to a polypeptide corresponding to a marker of the invention,preferably an antibody with a detectable label. Antibodies can bepolyclonal, or more preferably, monoclonal. An intact antibody, or afragment thereof, e.g., Fab or F(ab′) 2 can be used. The term “labeled”,with regard to the probe or antibody, is intended to encompassdirect-labeling of the probe or antibody by coupling, i.e., physicallylinking, a detectable substance to the probe or antibody, as well asindirect-labeling of the probe or antibody by reactivity with anotherreagent that is directly-labeled. Examples of indirect labeling includedetection of a primary antibody using a fluorescently-labeled secondaryantibody and end-labeling of a DNA probe with biotin such that it can bedetected with fluorescently-labeled streptavidin.

Proteins from individuals can be isolated using techniques that arewell-known to those of skill in the art. The protein isolation methodsemployed can, e.g., be such as those described in Harlow & Lane (1988),supra. A variety of formats can be employed to determine whether asample contains a protein that binds to a given antibody. Expression ofvarious biomarkers in a sample can be analyzed by a number ofmethodologies, many of which are known in the art and understood by theskilled artisan, including but not limited to, immunohistochemicaland/or Western analysis, quantitative blood based assays (as for exampleSerum ELISA) (to examine, for example, levels of protein expression),biochemical enzymatic activity assays, in situ hybridization, Northernanalysis and/or PCR analysis of mRNAs, as well as any one of the widevariety of assays that can be performed by gene and/or tissue arrayanalysis. Typical protocols for evaluating the status of genes and geneproducts are found, for example in Ausubel et al. eds., 1995, CurrentProtocols In Molecular Biology, Units 2 (Northern Blotting), 4 (SouthernBlotting), 15 (Immunoblotting) and 18 (PCR Analysis). A skilled artisancan readily adapt known protein/antibody detection methods for use indetermining whether cells express a marker of the present invention andthe relative concentration of that specific polypeptide expressionproduct in blood or other body tissues.

In such alternative methods, a sample may be contacted with an antibodyspecific for said biomarker under conditions sufficient for anantibody-biomarker complex to form, and then detecting said complex. Thepresence of the biomarker may be detected in a number of ways, such asby Western blotting and ELISA procedures for assaying a wide variety oftissues and samples, including plasma or serum. A wide range ofimmunoassay techniques using such an assay format are available, see,e.g., U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These includeboth single-site and two-site or “sandwich” assays of thenon-competitive types, as well as in the traditional competitive bindingassays. These assays also include direct binding of a labeled antibodyto a target biomarker.

Sandwich assays are among the most useful and commonly used assays. Anumber of variations of the sandwich assay technique exist, and all areintended to be encompassed by the present invention. Briefly, in atypical forward assay, an unlabelled antibody is immobilized on a solidsubstrate, and the sample to be tested brought into contact with thebound molecule. After a suitable period of incubation, for a period oftime sufficient to allow formation of an antibody-antigen complex, asecond antibody specific to the antigen, labeled with a reportermolecule capable of producing a detectable signal is then added andincubated, allowing time sufficient for the formation of another complexof antibody-antigen-labeled antibody. Any unreacted material is washedaway, and the presence of the antigen is determined by observation of asignal produced by the reporter molecule. The results may either bequalitative, by simple observation of the visible signal, or may bequantitated by comparing with a control sample containing known amountsof biomarker.

Variations on the forward assay include a simultaneous assay, in whichboth sample and labeled antibody are added simultaneously to the boundantibody. These techniques are well known to those skilled in the art,including any minor variations as will be readily apparent. In a typicalforward sandwich assay, a first antibody having specificity for thebiomarker is either covalently or passively bound to a solid surface.The solid surface is typically glass or a polymer, the most commonlyused polymers being cellulose, polyacrylamide, nylon, polystyrene,polyvinyl chloride or polypropylene. The solid supports may be in theform of tubes, beads, discs of microplates, or any other surfacesuitable for conducting an immunoassay. The binding processes arewell-known in the art and generally consist of cross-linking covalentlybinding or physically adsorbing, the polymer-antibody complex is washedin preparation for the test sample. An aliquot of the sample to betested is then added to the solid phase complex and incubated for aperiod of time sufficient (e.g. 2-40 minutes or overnight if moreconvenient) and under suitable conditions (e.g. from room temperature to40° C. such as between 25° C. and 32° C. inclusive) to allow binding ofany subunit present in the antibody. Following the incubation period,the antibody subunit solid phase is washed and dried and incubated witha second antibody specific for a portion of the biomarker. The secondantibody is linked to a reporter molecule which is used to indicate thebinding of the second antibody to the molecular marker.

An alternative method involves immobilizing the target biomarkers in thesample and then exposing the immobilized target to specific antibodywhich may or may not be labeled with a reporter molecule. Depending onthe amount of target and the strength of the reporter molecule signal, abound target may be detectable by direct labeling with the antibody.Alternatively, a second labeled antibody, specific to the first antibodyis exposed to the target-first antibody complex to form a target-firstantibody-second antibody tertiary complex. The complex is detected bythe signal emitted by the reporter molecule. By “reporter molecule”, asused in the present specification, is meant a molecule which, by itschemical nature, provides an analytically identifiable signal whichallows the detection of antigen-bound antibody. The most commonly usedreporter molecules in this type of assay are either enzymes,fluorophores or radionuclide containing molecules (i.e. radioisotopes)and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to thesecond antibody, generally by means of glutaraldehyde or periodate. Aswill be readily recognized, however, a wide variety of differentconjugation techniques exist, which are readily available to the skilledartisan. Commonly used enzymes include horseradish peroxidase, glucoseoxidase, -galactosidase and alkaline phosphatase, amongst others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. Examples of suitable enzymes include alkalinephosphatase and peroxidase. It is also possible to employ fluorogenicsubstrates, which yield a fluorescent product rather than thechromogenic substrates noted above. In all cases, the enzyme-labeledantibody is added to the first antibody-molecular marker complex,allowed to bind, and then the excess reagent is washed away. A solutioncontaining the appropriate substrate is then added to the complex ofantibody-antigen-antibody. The substrate will react with the enzymelinked to the second antibody, giving a qualitative visual signal, whichmay be further quantitated, usually spectrophotometrically, to give anindication of the amount of biomarker which was present in the sample.Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody adsorbs the light energy,inducing a state to excitability in the molecule, followed by emissionof the light at a characteristic color visually detectable with a lightmicroscope. As in the EIA, the fluorescent labeled antibody is allowedto bind to the first antibody-molecular marker complex. After washingoff the unbound reagent, the remaining tertiary complex is then exposedto the light of the appropriate wavelength, the fluorescence observedindicates the presence of the molecular marker of interest.Immunofluorescence and EIA techniques are both very well established inthe art. However, other reporter molecules, such as radioisotope,chemiluminescent or bioluminescent molecules, may also be employed.

Methods of the invention further include protocols which examine thepresence and/or expression of mRNAs, in a tissue or cell sample. Methodsfor the evaluation of mRNAs in cells are well known and include, forexample, hybridization assays using complementary DNA probes (such as insitu hybridization using labeled riboprobes, Northern blot and relatedtechniques) and various nucleic acid amplification assays (such asRT-PCR and other amplification type detection methods, such as, forexample, branched DNA, SISBA, TMA and the like).

In an embodiment, the level of mRNA corresponding to the marker can bedetermined both by in situ and by in vitro formats in a biologicalsample using methods known in the art. Many expression detection methodsuse isolated RNA. For in vitro methods, any RNA isolation technique thatdoes not select against the isolation of mRNA can be utilized for thepurification of RNA from cells. See, e.g., Ausubel et al., Ed., Curr.Prot. Mol. Biol., John Wiley & Sons, NY (1987-1999). Additionally, largenumbers of tissue samples can readily be processed using techniqueswell-known to those of skill in the art, such as, e.g., the single-stepRNA isolation process of U.S. Pat. No. 4,843,155. The isolated mRNA canbe used in hybridization or amplification assays that include, but arenot limited to, Southern or Northern analyses, PCR analyses and probearrays. One preferred diagnostic method for the detection of mRNA levelsinvolves contacting the isolated mRNA with a nucleic acid molecule(probe) that can hybridize to the mRNA encoded by the gene beingdetected. The nucleic acid probe can be, e.g., a full-length cDNA, or aportion thereof, such as an oligonucleotide of at least 7, 15, 30, 50,100, 250 or 500 nucleotides in length and sufficient to specificallyhybridize under stringent conditions to a mRNA or genomic DNA encoding amarker of the present invention. Other suitable probes for use in thediagnostic assays of the invention are described herein. Hybridizationof an mRNA with the probe indicates that the marker in question is beingexpressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example, by running the isolated mRNA on an agarosegel and transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in an Affymetrix gene chip array. A skilled artisan can readilyadapt known mRNA detection methods for use in detecting the level ofmRNA encoded by the markers of the present invention.

Although amplification of molecules is not required in the presentinvention as discussed in the examples section, one of skill in the artcould use amplification methods. One alternative method for determiningthe level of mRNA corresponding to a marker of the present invention ina sample involves the process of nucleic acid amplification, e.g., byRT-PCR (the experimental embodiment set forth in Mullis, U.S. Pat. No.4,683,202 (1987); ligase chain reaction, self-sustained sequencereplication, Guatelli et al., Proc. Natl. Acad. Sci. USA, Vol. 87, pp.1874-1878 (1990); transcriptional amplification system, Kwoh et al.,Proc. Natl. Acad. Sci. USA, Vol. 86, pp. 1173-1177 (1989); Q-BetaReplicase, Lizardi et al., Biol. Technology, Vol. 6, p. 1197 (1988);rolling circle replication, U.S. Pat. No. 5,854,033 (1988); or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well-known to those of skill in theart. These detection schemes are especially useful for the detection ofthe nucleic acid molecules if such molecules are present in very lownumbers. As used herein, amplification primers are defined as being apair of nucleic acid molecules that can anneal to 5′ or 3′ regions of agene (plus and minus strands, respectively, or vice-versa) and contain ashort region in between. In general, amplification primers are fromabout 10-30 nucleotides in length and flank a region from about 50-200nucleotides in length. Under appropriate conditions and with appropriatereagents, such primers permit the amplification of a nucleic acidmolecule comprising the nucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated form the cellsprior to detection. In such methods, a cell or tissue sample isprepared/processed using known histological methods. The sample is thenimmobilized on a support, typically a glass slide, and then contactedwith a probe that can hybridize to mRNA that encodes the marker.

As an alternative to making determinations based on the absoluteexpression level of the marker, determinations may be based on thenormalized expression level of the marker. Expression levels arenormalized by correcting the absolute expression level of a marker bycomparing its expression to the expression of a gene that is not amarker, e.g., a housekeeping gene that is constitutively expressed.Suitable genes for normalization include housekeeping genes, such as theactin gene or epithelial cell-specific genes. This normalization allowsthe comparison of the expression level in one sample, e.g., a patientsample, to another sample or between samples from different sources.

Alternatively, the expression level can be provided as a relativeexpression level. To determine a relative expression level of a marker,the level of expression of the marker is determined for 10 or moresamples of normal versus disease biological samples, preferably 50 ormore samples, prior to the determination of the expression level for thesample in question. The mean expression level of each of the genesassayed in the larger number of samples is determined and this is usedas a baseline expression level for the marker. The expression level ofthe marker determined for the test sample (absolute level of expression)is then divided by the mean expression value obtained for that marker.This provides a relative expression level.

Preferably, the samples used in the baseline determination will be frompatients who do not have the polymorphism. The choice of the cell sourceis dependent on the use of the relative expression level. Usingexpression found in normal tissues as a mean expression score aids invalidating whether the marker assayed is specific (versus normal cells).In addition, as more data is accumulated, the mean expression value canbe revised, providing improved relative expression values based onaccumulated data.

Antibodies and Aptamers

In a preferred embodiment, the antibodies and aptamers specifically bindeach component of the biomarkers described herein. The componentsinclude the nucleic acid sequences, complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof of eachcomponent in each biomarker.

Aptamer polynucleotides are typically single-stranded standardphosphodiester DNA (ssDNA). Close DNA analogs can also be incorporatedinto the aptamer as described below.

A typical aptamer discovery procedure is described below:

A polynucleotide comprising a randomized sequence between “arms” havingconstant sequence is synthesized. The arms can include restriction sitesfor convenient cloning and can also function as priming sites for PCRprimers. The synthesis can easily be performed on commercialinstruments.

The target protein is treated with the randomized polynucleotide. Thetarget protein can be in solution and then the complexes immobilized andseparated from unbound nucleic acids by use of an antibody affinitycolumn. Alternatively, the target protein might be immobilized beforetreatment with the randomized polynucleotide.

The target protein-polynucleotide complexes are separated from theuncomplexed material and then the bound polynucleotides are separatedfrom the target protein. The bound nucleic acid can then becharacterized, but is more commonly amplified, e.g. by PCR and thebinding, separation and amplification steps are repeated. In manyinstances, use of conditions increasingly promoting separation of thenucleic acid from the target protein, e.g. higher salt concentration, inthe binding buffer used in step 2) in subsequent iterations, results inidentification of polynucleotides having increasingly high affinity forthe target protein.

The nucleic acids showing high affinity for the target proteins areisolated and characterized. This is typically accomplished by cloningthe nucleic acids using restriction sites incorporated into the arms,and then sequencing the cloned nucleic acid:

The affinity of aptamers for their target proteins is typically in thenanomolar range, but can be as low as the picomolar range. That is K_(D)is typically 1 pM to 500 nM, more typically from 1 pM to 100 nM.Apatmers having an affinity of K_(D) in the range of 1 pM to 10 nM arealso useful.

Aptamer polynucleotides can be synthesized on a commercially availablenucleic acid synthesizer by methods known in the art. The product can bepurified by size selection or chromatographic methods.

Aptamer polynucleotides are typically from about 10 to 200 nucleotideslong, more typically from about 10 to 100 nucleotides long, still moretypically from about 10 to 50 nucleotides long and yet more typicallyfrom about 10 to 25 nucleotides long. A preferred range of length isfrom about 10 to 50 nucleotides.

The aptamer sequences can be chosen as a desired sequence, or random orpartially random populations of sequences can be made and then selectedfor specific binding to a desired target protein by assay in vitro. Anyof the typical nucleic acid-protein binding assays known in the art canbe used, e.g. “Southwestern” blotting using either labeledoligonucleotide or labeled protein as the probe. See also U.S. Pat. No.5,445,935 for a fluorescence polarization assay of protein-nucleic acidinteraction.

Appropriate nucleotides for aptamer synthesis and their use, andreagents for covalent linkage of proteins to nucleic acids and theiruse, are considered known in the art.

A desired aptamer-protein complex, for example, aptamer-thrombin complexof the invention can be labeled and used as a diagnostic agent in vitroin much the same manner as any specific protein-binding agent, e.g. amonoclonal antibody. Thus, an aptamer-protein complex of the inventioncan be used to detect and quantitate the amount of its target protein ina sample, e.g. a blood sample, to provide diagnosis of a disease statecorrelated with the amount of the protein in the sample.

A desired aptamer-target/bait molecular complex can also be used fordiagnostic imaging. In imaging uses, the complexes are labeled so thatthey can be detected outside the body. Typical labels are radioisotopes,usually ones with short half-lives. The usual imaging radioisotopes,such as ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ^(99m)TC, ¹⁸⁶Re, ¹⁸⁸Re, ⁶⁴Cu, ⁶⁷Cu,²¹²Bi, ²¹³Bi, ⁶⁷Ga, ⁹⁰Y, ¹¹¹In, ¹⁸F, ³H, ¹⁴C, ³⁵S or ³²P can be used.Nuclear magnetic resonance (NMR) imaging enhancers, such asgadolinium-153, can also be used to label the complex for detection byNMR. Methods and reagents for performing the labeling, either in thepolynucleotide or in the protein moiety, are considered known in theart.

In a preferred embodiment, an antibody or aptamer is specific for eachgene sequence of the biomarker comprising a biomarker comprises genesequences: 232669_at (Hypoxia inducible factor 3, alpha subunit),214951_at (solute carrier family 26, member 10), 243482_at (Epidermalgrowth factor receptor pathway substrate 15-like 1), 226210_s_at(maternally expressed 3), 232159_at (Epidermal growth factor receptorpathway substrate 15-like 1), 233026_s_at (PDZ domain containing),211996_s_at (KIAA0220-like protein hypothetical gene LOC 283846),243774_at (mucin 20, cell surface associated), 242551_at (Chromosome 18open reading frame), 244548_at (Rho GTPase activating protein 26),244208_at (Checkpoint suppressor 1), 239984_at (Sodium channel,voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis, cloneADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof.

In a preferred embodiment, an antibody or aptamer is specific for eachgene sequence of the biomarker comprising a biomarker comprises genesequences: 1558458_at (Hypothetical LOC401320), 1560049_at (CUG tripletrepeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNA binding motifprotein 5, RBM5), 201655_s_at (heparan sulfate proteoglycan 2(perlecan), HSPG2), 202379_s_at (natural killer-tumor recognitionsequence, NKTR), 202808_at, 203071_at (sema domain, immunoglobulindomain (Ig), short basic domain, secreted, (semaphorin) 3B, SEMA3B),203748_x_at (RNA binding motif, single stranded interacting protein 1,RBMS1), 203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4,ABCD4), 204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7,MYH6///MYH7), 204978_at (splicing factor, arginine/serine-rich 16,SFRS16), 206209_s_at (carbonic anhydrase IV, CA4), 207541_s_at (exosomecomponent 10, EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at(cysteine-rich protein 2, CRIP2), 209354_at (tumor necrosis factorreceptor superfamily, member 14 TNFRSF14), 210628_x_at (latenttransforming growth factor beta binding protein 4, LTBP4), 211909_x_at(prostaglandin E receptor 3 (subtype EP3), PTGER3), 211996_at(KIAA0220-like protein, nuclear pore complex (LOC23117), 212487_at (Gpatch domain containing 8, GPATCH8), 213946_s_at (obscurin-like 1,OBSL1), 214951_at (solute carrier family 26, member 10, SLC26A10),220219_s_at (leucine rich repeat containing 37A, LRRC37A), 221071_at,221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27),221806_s_at (SET domain containing 5, SETD5), 221833_at (Lon peptidase2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S. cerevisiae), LUC7L),224260_at (CDNA clone IMAGE:4478733), 225562_at (AS p21 proteinactivator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156 (from cloneDKFZp762N156), 227968_at (Parkinson disease 7 domain containing 1,PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9, MRPS9),229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis, cloneADKA03430), 238185_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 241597_at (Arginine-glutamic acid dipeptide (RE)repeats, RERE), 242551_at (Chromosome 18 open reading frame 1, C18orf1),244208_at (Checkpoint suppressor 1, CHES1), 244494_at (Zinc finger,DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPase activatingprotein 26, ARHGAP26) complementary sequences, fragments, alleles,variants, derivatives and/or gene products thereof.

Drug Discovery

In other preferred embodiments, the molecular signatures are useful forthe identification of new drugs in the treatment of cardiovasculardiseases and disorders.

Small Molecules:

Small molecule test compounds or candidate therapeutic compounds caninitially be members of an organic or inorganic chemical library. Asused herein, “small molecules” refers to small organic or inorganicmolecules of molecular weight below about 3,000 Daltons. The smallmolecules can be natural products or members of a combinatorialchemistry library. A set of diverse molecules should be used to cover avariety of functions such as charge, aromaticity, hydrogen bonding,flexibility, size, length of side chain, hydrophobicity, and rigidity.Combinatorial techniques suitable for synthesizing small molecules areknown in the art, e.g., as exemplified by Obrecht and Villalgordo,Solid-Supported Combinatorial and Parallel Synthesis ofSmall-Molecular-Weight Compound Libraries, Pergamon-Elsevier ScienceLimited (1998), and include those such as the “split and pool” or“parallel” synthesis techniques, solid-phase and solution-phasetechniques, and encoding techniques (see, for example, Czarnik, Curr.Opin. Chem. Bio., 1:60 (1997). In addition, a number of small moleculelibraries are commercially available.

Particular screening applications of this invention relate to thetesting of pharmaceutical compounds in drug research. The reader isreferred generally to the standard textbook “In vitro Methods inPharmaceutical Research”, Academic Press, 1997, and U.S. Pat. No.5,030,015). Assessment of the activity of candidate pharmaceuticalcompounds generally involves administering a candidate compound,determining any change in the morphology, marker phenotype andexpression, or metabolic activity of the cells and function of the cellsthat is attributable to the compound (compared with untreated cells orcells treated with an inert compound), and then correlating the effectof the compound with the observed change.

The screening may be done, for example, either because the compound isdesigned to have a pharmacological effect on certain cell types, orbecause a compound designed to have effects elsewhere may haveunintended side effects. Two or more drugs can be tested in combination(by combining with the cells either simultaneously or sequentially), todetect possible drug-drug interaction effects. In some applications,compounds are screened initially for potential toxicity (Castell et al.,pp. 375-410 in “In vitro Methods in Pharmaceutical Research,” AcademicPress, 1997). Cytotoxicity can be determined in the first instance bythe effect on cell viability, survival, morphology, and expression orrelease of certain markers, receptors or enzymes. Effects of a drug onchromosomal DNA can be determined by measuring DNA synthesis or repair.[³H]thymidine or BrdU incorporation, especially at unscheduled times inthe cell cycle, or above the level required for cell replication, isconsistent with a drug effect. Unwanted effects can also include unusualrates of sister chromatid exchange, determined by metaphase spread. Thereader is referred to A. Vickers (PP 375-410 in “In vitro Methods inPharmaceutical Research,” Academic Press, 1997) for further elaboration.

In one embodiment of the invention, a method of identifying a candidateagent is provided said method comprising: (a) contacting a biologicalsample from a patient with the candidate agent and determining the levelof expression of one or more biomarkers described herein; (b)determining the level of expression of a corresponding biomarker orbiomarkers in an aliquot of the biological sample not contacted with thecandidate agent; (c) observing the effect of the candidate agent bycomparing the level of expression of the biomarker or biomarkers in thealiquot of the biological sample contacted with the candidate agent andthe level of expression of the corresponding biomarker or biomarkers inthe aliquot of the biological sample not contacted with the candidateagent; and (d) identifying said agent from said observed effect, whereinan at least 10% difference between the level of expression of thebiomarker gene or combination of biomarker genes in the aliquot of thebiological sample contacted with the candidate agent and the level ofexpression of the corresponding biomarker gene or combination ofbiomarker genes in the aliquot of the biological sample not contactedwith the candidate agent is an indication of an effect of the candidateagent.

In preferred embodiments, the effects of the drug are correlated withthe expression of the molecular signatures associated with a goodprognosis as described in detail in the examples which follow.

In another embodiment of the invention, a candidate agent derived by themethod according to the invention is provided.

In another embodiment of the invention, a pharmaceutical preparationcomprising an agent according to the invention is provided.

In another preferred embodiment of the invention, a method of producinga drug comprising the steps of the method according to the invention (i)synthesizing the candidate agent identified in step (c) above or ananalog or derivative thereof in an amount sufficient to provide saiddrug in a therapeutically effective amount to a subject; and/or (ii)combining the drug candidate the candidate agent identified in step (c)above or an analog or derivative thereof with a pharmaceuticallyacceptable carrier.

Vectors, Cells:

In some embodiments it is desirable to express the biomolecules thatcomprise a biomarker, in a vector and in cells. The applications of suchcombinations are unlimited. The vectors and cells expressing the one ormore biomolecules can be used in assays, kits, drug discovery,diagnostics, prognostics and the like. The cells can be stem cellsisolated from the bone marrow as a progenitor cell, or cells obtainedfrom any other source, such as for example, ATCC.

“Bone marrow derived progenitor cell” (BMDC) or “bone marrow derivedstem cell” refers to a primitive stem cell with the machinery forself-renewal constitutively active. Included in this definition are stemcells that are totipotent, pluripotent and precursors. A “precursorcell” can be any cell in a cell differentiation pathway that is capableof differentiating into a more mature cell. As such, the term “precursorcell population” refers to a group of cells capable of developing into amore mature cell. A precursor cell population can comprise cells thatare totipotent, cells that are pluripotent and cells that are stem celllineage restricted (i.e. cells capable of developing into less than allhematopoietic lineages, or into, for example, only cells of erythroidlineage). As used herein, the term “totipotent cell” refers to a cellcapable of developing into all lineages of cells. Similarly, the term“totipotent population of cells” refers to a composition of cellscapable of developing into all lineages of cells. Also as used herein,the term “pluripotent cell” refers to a cell capable of developing intoa variety (albeit not all) lineages and are at least able to developinto all hematopoietic lineages (e.g., lymphoid, erythroid, andthrombocytic lineages). Bone marrow derived stem cells contain twowell-characterized types of stem cells. Mesenchymal stem cells (MSC)normally form chondrocytes and osteoblasts. Hematopoietic stem cells(HSC) are of mesodermal origin that normally give rise to cells of theblood and immune system (e.g., erythroid, granulocyte/macrophage,magakaryocite and lymphoid lineages). In addition, hematopoietic stemcells also have been shown to have the potential to differentiate intothe cells of the liver (including hepatocytes, bile duct cells), lung,kidney (e.g., renal tubular epithelial cells and renal parenchyma),gastrointestinal tract, skeletal muscle fibers, astrocytes of the CNS,Purkinje neurons, cardiac muscle (e.g., cardiomyocytes), endothelium andskin.

In a preferred embodiment, a method of identifying candidate therapeuticcompounds comprises culturing cells expressing at least one biomoleculeselected from: 232669_at (Hypoxia inducible factor 3, alpha subunit),214951_at (solute carrier family 26, member 10), 243482_at (Epidermalgrowth factor receptor pathway substrate 15-like 1), 226210_s_at(maternally expressed 3), 232159_at (Epidermal growth factor receptorpathway substrate 15-like 1), 233026_s_at (PDZ domain containing),211996_s_at (KIAA0220-like protein hypothetical gene LOC 283846),243774_at (mucin 20, cell surface associated), 242551_at (Chromosome 18open reading frame), 244548_at (Rho GTPase activating protein 26),244208_at (Checkpoint suppressor 1), 239984_at (Sodium channel,voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis, cloneADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at (EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific) complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof, with acandidate therapeutic agent; identifying candidate therapeutic agentswhich modulate the expression of the biomolecules and identifying acandidate therapeutic agent. Preferably, a candidate therapeutic agentcomprises organic molecules, inorganic molecules, vaccines, antibodies,nucleic acid molecules, proteins, peptides and vectors expressingnucleic acid molecules.

In a preferred embodiment, a method of identifying candidate therapeuticcompounds comprises culturing cells expressing at least one biomoleculeselected from: 1558458_at (Hypothetical LOC401320), 1560049_at (CUGtriplet repeat, RNA binding protein 2, CUGBP2), 201394_s_at (RNA bindingmotif protein 5, RBM5), 201655_s_at (heparan sulfate proteoglycan 2(perlecan), HSPG2), 202379_s_at (natural killer-tumor recognitionsequence, NKTR), 202808_at, 203071_at (sema domain, immunoglobulindomain (Ig), short basic domain, secreted, (semaphorin) 3B, SEMA3B),203748_x_at (RNA binding motif, single stranded interacting protein 1,RBMS1), 203981_s_at (ATP-binding cassette, sub-family D (ALD), member 4,ABCD4), 204737_s_at (myosin, heavy chain 6, myosin, heavy chain 7,MYH6///MYH7), 204978_at (splicing factor, arginine/serine-rich 16,SFRS16), 206209_s_at (carbonic anhydrase IV, CA4), 207541_s_at (exosomecomponent 10, EXOSC10), 207798_s_at (ataxin 2-like, ATXN2L), 208978_at(cysteine-rich protein 2, CRIP2), 209354_at (tumor necrosis factorreceptor superfamily, member 14 TNFRSF14), 210628_x_at (latenttransforming growth factor beta binding protein 4, LTBP4), 211909_x_at(prostaglandin E receptor 3 (subtype EP3), PTGER3), 211996_at(KIAA0220-like protein, nuclear pore complex (LOC23117), 212487_at (Gpatch domain containing 8, GPATCH8), 213946_s_at (obscurin-like 1,OBSL1), 214951_at (solute carrier family 26, member 10, SLC26A10),220219_s_at (leucine rich repeat containing 37A, LRRC37A), 221071_at,221780_s_at (DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27),221806_s_at (SET domain containing 5, SETD5), 221833_at (Lon peptidase2, peroxisomal, LONP2), 223546_x_at (LUC7-like (S. cerevisiae), LUC7L),224260_at (CDNA clone IMAGE:4478733), 225562_at (AS p21 proteinactivator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156 (from cloneDKFZp762N156), 227968_at (Parkinson disease 7 domain containing 1,PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9, MRPS9),229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis, cloneADKA03430), 238185_at (RNA binding motif, single stranded interactingprotein 1, RBMS1), 241597_at (Arginine-glutamic acid dipeptide (RE)repeats, RERE), 242551_at (Chromosome 18 open reading frame 1, C18orf1),244208_at (Checkpoint suppressor 1, CHES1), 244494_at (Zinc finger,DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPase activatingprotein 26, ARHGAP26) complementary sequences, fragments, alleles,variants, derivatives and/or gene products thereof, with a candidatetherapeutic agent; identifying candidate therapeutic agents whichmodulate the expression of the biomolecules and identifying a candidatetherapeutic agent. Preferably, a candidate therapeutic agent comprisesorganic molecules, inorganic molecules, vaccines, antibodies, nucleicacid molecules, proteins, peptides and vectors expressing nucleic acidmolecules.

Such compounds are useful, e.g., as candidate therapeutic compounds forthe treatment of heart disease, heart disorders and conditions thereof.Thus, included herein are methods for screening for candidatetherapeutic compounds for the treatment of, for example, myocarditis,Coronary Heart Disease, angina, Acute Coronary Syndrome, Aortic Aneurysmand Dissection, arrhythmias, Cardiomyopathy, Congenital Heart Disease,congestive heart failure or chronic heart failure, pericarditis, and thelike. The methods include administering the compound to a model of thecondition, e.g., contacting a cell (in vitro) model with the compound,or administering the compound to an animal model of the condition, e.g.,an animal model of a condition associated with heart disease. The modelis then evaluated for an effect of the candidate compound on theclinical outcome in the model and can be considered a candidatetherapeutic compound for the treatment of the condition. Such effectscan include clinically relevant effects, decreased pain; increased lifespan; and so on. Such effects can be determined on a macroscopic ormicroscopic scale. Candidate therapeutic compounds identified by thesemethods can be further verified, e.g., by administration to humansubjects in a clinical trial.

The biomolecules can be expressed from one or more vectors. A “vector”(sometimes referred to as gene delivery or gene transfer “vehicle”)refers to a macromolecule or complex of molecules comprising apolynucleotide to be delivered to a host cell, either in vitro or invivo. The polynucleotide to be delivered may comprise a coding sequenceof interest in gene therapy. Vectors include, for example, viral vectors(such as adenoviruses (“Ad”), adeno-associated viruses (AAV), andretroviruses), liposomes and other lipid-containing complexes, and othermacromolecular complexes capable of mediating delivery of apolynucleotide to a host cell. Vectors can also comprise othercomponents or functionalities that further modulate gene delivery and/orgene expression, or that otherwise provide beneficial properties to thetargeted cells. As described and illustrated in more detail below, suchother components include, for example, components that influence bindingor targeting to cells (including components that mediate cell-type ortissue-specific binding); components that influence uptake of the vectornucleic acid by the cell; components that influence localization of thepolynucleotide within the cell after uptake (such as agents mediatingnuclear localization); and components that influence expression of thepolynucleotide. Such components also might include markers, such asdetectable and/or selectable markers that can be used to detect orselect for cells that have taken up and are expressing the nucleic aciddelivered by the vector. Such components can be provided as a naturalfeature of the vector (such as the use of certain viral vectors whichhave components or functionalities mediating binding and uptake), orvectors can be modified to provide such functionalities. Other vectorsinclude those described by Chen et al; BioTechniques, 34: 167-171(2003). A large variety of such vectors are known in the art and aregenerally available.

Kits

In another preferred embodiment, a kit is provided comprising any one ormore of the biomolecules comprising: 232669_at (Hypoxia inducible factor3, alpha subunit), 214951_at (solute carrier family 26, member 10),243482_at (Epidermal growth factor receptor pathway substrate 15-like1), 226210_s_at (maternally expressed 3), 232159_at (Epidermal growthfactor receptor pathway substrate 15-like 1), 233026_s_at (PDZ domaincontaining), 211996_s_at (KIAA0220-like protein hypothetical gene LOC283846), 243774_at (mucin 20, cell surface associated), 242551_at(Chromosome 18 open reading frame), 244548_at (Rho GTPase activatingprotein 26), 244208_at (Checkpoint suppressor 1), 239984_at (Sodiumchannel, voltage-gated, type VII, alpha), 230683_at (CDNA:FLJ20892 fis,clone ADKA03430), 214869_at (apolipoprotein L, 6), 241597_at(Arginine-glutamic acid dipeptide (RE) repeats), 235887_at (Smg-6homolog, nonsense mediated mRNA decay factor (C. elegans)), 229957_at(transmembrane protein 91), 223546_x_at (LUC7L-like (S. cerevisiae)),239567_at (Rho GTPase activating protein 10), 242194_at (Cullin 4A),1558525_at (hypothetical protein LOC283901), 227178_at (CUG tripletrepeat, RNA binding protein 2), 228198_s_at (Mitochondrial ribosomalprotein S9), 202379_s_at (natural killer-tumor recognition sequence),224260_at (CDNA clone), 238643_at (Neuroblastoma, suppression oftumorigenicity 1), 232253_at (RAD50 homolog (S. cerevisiae)), 227968_at(Parkinson disease 7 domain containing 1), 233197_at (kelch-like 9(Drosophila)), 244512_at (transcribed locus strongly similar toXP_0010813421), 233443_at (hypothetical protein LOC389362), 231275_at(FLJ42875 protein), 226419_s_at (hypothetical protein LOC64546),201221_s_at small nuclear ribonucleoprotein 70 kDa polypeptide),209354_at (tumor necrosis factor receptor family member 14), 226571_s_at(protein tyrosine phosphatase receptor type, S), 220728_at S(EST),203071_at (sema domain, immunoglobulin domain (Ig), short basic domain),213946_s_at obscurin-like 1, similar to titin isoform N2-B), 201394_s_at(RNA binding motif protein 5), 203748_x_at (RNA binding motif, singlestranded interacting protein 1), 223147_s_at (WD repeat domain 33),213773_x_at (NOL/NOP2/Sun domain family, member 5), 1560049_at CUGtriplet repeat, RNA binding protein 2), 243974_at (CDNA cloneIMAGE:4821815), 201510_at E74-like factor 3 (ets domain transcriptionfactor, epithelial specific), complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof.

In another preferred embodiment, a kit is provided comprising any one ormore of the biomolecules comprising: 1558458_at (HypotheticalLOC401320), 1560049_at (CUG triplet repeat, RNA binding protein 2,CUGBP2), 201394_s_at (RNA binding motif protein 5, RBM5), 201655_s_at(heparan sulfate proteoglycan 2 (perlecan), HSPG2), 202379_s_at (naturalkiller-tumor recognition sequence, NKTR), 202808_at, 203071_at (semadomain, immunoglobulin domain (Ig), short basic domain, secreted,(semaphorin) 3B, SEMA3B), 203748_x_at (RNA binding motif, singlestranded interacting protein 1, RBMS1), 203981_s_at (ATP-bindingcassette, sub-family D (ALD), member 4, ABCD4), 204737_s_at (myosin,heavy chain 6, myosin, heavy chain 7, MYH6///MYH7), 204978_at (splicingfactor, arginine/serine-rich 16, SFRS16), 206209_s_at (carbonicanhydrase IV, CA4), 207541_s_at (exosome component 10, EXOSC10),207798_s_at (ataxin 2-like, ATXN2L), 208978_at (cysteine-rich protein 2,CRIP2), 209354_at (tumor necrosis factor receptor superfamily, member 14TNFRSF14), 210628_x_at (latent transforming growth factor beta bindingprotein 4, LTBP4), 211909_x_at (prostaglandin E receptor 3 (subtypeEP3), PTGER3), 211996_at (KIAA0220-like protein, nuclear pore complex(LOC23117), 212487_at (G patch domain containing 8, GPATCH8),213946_s_at (obscurin-like 1, OBSL1), 214951_at (solute carrier family26, member 10, SLC26A10), 220219_s_at (leucine rich repeat containing37A, LRRC37A), 221071_at, 221780_s_at (DEAD (Asp-Glu-Ala-Asp) boxpolypeptide 27DDX27), 221806_s_at (SET domain containing 5, SETD5),221833_at (Lon peptidase 2, peroxisomal, LONP2), 223546_x_at (LUC7-like(S. cerevisiae), LUC7L), 224260_at (CDNA clone IMAGE:4478733), 225562_at(AS p21 protein activator 3, RASA3), 226040_at (MRNA; cDNA DKFZp762N156(from clone DKFZp762N156), 227968_at (Parkinson disease 7 domaincontaining 1, PDDC1), 228198_s_at (Mitochondrial ribosomal protein S9,MRPS9), 229830_at (Transcribed locus), 230683_at (CDNA: FLJ20892 fis,clone ADKA03430), 238185_at (RNA binding motif, single strandedinteracting protein 1, RBMS1), 241597_at (Arginine-glutamic aciddipeptide (RE) repeats, RERE), 242551_at (Chromosome 18 open readingframe 1, C18orf1), 244208_at (Checkpoint suppressor 1, CHES1), 244494_at(Zinc finger, DHHC-type containing 1, ZDHHC1), and 244548_at (Rho GTPaseactivating protein 26, ARHGAP26), complementary sequences, fragments,alleles, variants, derivatives and/or gene products thereof.

For use in the applications described or suggested above, kits orarticles of manufacture are also provided by the invention. Such kitsmay comprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe that is or can be detectably labeled. Wherethe kit utilizes nucleic acid hybridization to detect the target nucleicacid, the kit may also have containers containing nucleotide(s) foramplification of the target nucleic acid sequence and/or a containercomprising a reporter-means, such as a biotin-binding protein, such asavidin or streptavidin, bound to a reporter molecule, such as anenzymatic, florescent, or radioisotope label.

The kit of the invention will typically comprise the container describedabove and one or more other containers comprising materials desirablefrom a commercial and user standpoint, including buffers, diluents,filters, needles, syringes, and package inserts with instructions foruse. A label may be present on the container to indicate that thecomposition is used for a specific therapy or non-therapeuticapplication, and may also indicate directions for either in vivo or invitro use, such as those described above.

The kits of the invention have a number of embodiments. A typicalembodiment is a kit comprising a container, a label on said container,and a composition contained within said container, wherein thecomposition includes a primary antibody that binds to the biomoleculesof each molecular signature and instructions for using the antibody forevaluating the presence of biomolecules in at least one type ofmammalian cell. The kit can further comprise a set of instructions andmaterials for preparing a tissue sample and applying antibody and probeto the same section of a tissue sample. The kit may include both aprimary and secondary antibody, wherein the secondary antibody isconjugated to a label, e.g., an enzymatic label.

Another embodiment is a kit comprising a container, a label on saidcontainer, and a composition contained within said container, whereinthe composition includes a polynucleotide that hybridizes to acomplement of the polynucleotides under stringent conditions, the labelon said container indicates that the composition can be used to evaluatethe presence of a molecular signature in at least one type of mammaliancell, and instructions for using the polynucleotide for evaluating thepresence of biomolecule RNA or DNA in at least one type of mammaliancell.

Other optional components in the kit include, microarrays, one or morebuffers (e.g., block buffer, wash buffer, substrate buffer, etc), otherreagents such as substrate (e.g., chromogen) which is chemically alteredby an enzymatic label, epitope retrieval solution, control samples(positive and/or negative controls), control slide(s) etc.

All documents mentioned herein are incorporated herein by reference. Allpublications and patent documents cited in this application areincorporated by reference for all purposes to the same extent as if eachindividual publication or patent document were so individually denoted.By their citation of various references in this document, Applicants donot admit any particular reference is “prior art” to their invention.Embodiments of inventive compositions and methods are illustrated in thefollowing examples.

EXAMPLES

The following non-limiting Examples serve to illustrate selectedembodiments of the invention. It will be appreciated that variations inproportions and alternatives in elements of the components shown will beapparent to those skilled in the art and are within the scope ofembodiments of the present invention.

Example 1: Transcriptomic Biomarkers for Individual Risk Assessment inNew Onset Heart Failure

Accurate risk assessment and prediction of prognosis at firstpresentation are crucial for appropriate allocation of therapy,monitoring and patient management. In this study, a transcriptomic basedbiomarker (TBB) is presented which has been derived from a singleendomyocardial biopsy (EMB) and which predicts the long term clinicaloutcome of patients with idiopathic dilated cardiomyopathy (IDCM) withvery high accuracy.

Materials and Methods

Abbreviations:

NYHA, New York Heart Association classification; LV EF, left ventricularejection fraction, LVIDD, left ventricular internal diastolic dimension;PAP, pulmonary artery pressure; PCWP, pulmonary capillary wedgepressure.

Patients:

EMBs were collected from patients that were referred to Johns HopkinsHospital between 1997 and 2006 for evaluation of cardiomyopathy(n=35012). A clinical data base of patient outcome was maintainedconcurrently for a 10 year period beginning in 1997. All patients gavewritten informed consent for sample collection and medical chartabstraction. Transvenous EMBs from the right ventricular septum wereobtained for microarray analysis as previously described (Felker G M,Thompson R E, Hare J M et al. N Engl J Med 2000; 342: 1077-8). In orderto avoid possible disease specific confounding factors, samples from onesingle frequent type of cardiomyopathy (IDCM), were selected. IDCM was adiagnosis of exclusion after extensive histological work-up without anydetectable pathological signs. Within a repository of 180 IDCM samples,biopsies were selected in a case-control fashion based on the phenotypicextremes in survival of the cohort. A group with good prognosis (GP,n=25), was defined as having event free survival for at least 5 yearsafter initial presentation with heart failure symptoms; a group with badprognosis (BP, n=18), experienced an event within the first 2 years.Events included death (n=14), requirement for left ventricular assistdevice (n=2) or cardiac transplantation (n=2).

Processing of Biopsies:

EMBs were immediately flash frozen in liquid nitrogen for storage in abiorepository. All steps of RNA isolation and processing were performedaccording to MIAME guidelines (Minimum Information about a MicroarrayExperiment). Tissue samples (average diameter −2 mm) were homogenizedwith the MM 301 Mixer Mill (Retsch, Cat. No. 85120). Trizol reagenttogether with the Micro-to-Midi Total RNA Purification System(Invitrogen, Cat. No. 12183-018) was used for extraction of total RNA(success rate: 97% of samples). Concentration and integrity of total RNAwas measured with the Agilent 2100 Bioanalyzer. All RNA samplesexhibited intact 28S and 18S ribosomal RNA on denaturing agarose gelelectrophoresis and the 260/280 nm absorbance readings fell in theacceptable range of 1.8-2.1. An average amount of 586 ng total RNA wasisolated and preprocessed with the Ovation Biotin RNA Amplification andLabeling System (NuGen, Cat. No. 2300-12).

Microarray Hybridization:

Samples were hybridized to the Human Genome U133 Plus 2.0 Array fromAffymetrix without additional amplification step. The microarrayexperiments were judged successful when RNA isolation and microarrayhybridization met all the indices of quality control as specified in theAffymetrix Guideline for Assessing Sample and Array Quality. Averagebackground and noise of all chips registered within acceptable rangesand hybridization efficiencies were similar for all samples.

Statistical Analysis:

Raw intensity values from microarray hybridization were normalized withRobust Multiarray Average (RMA) implemented in the R package forstatistical computing (available at www.R-project.com) In the next step,Significance Analysis of Microarrays (SAM) was used to identifyphenotype specific differences in gene expression. SAM definessignificance with the q-value, an adjusted p-value for multiplecomparisons (Storey J. Journal of the Royal Statistical Society2007:64:479-498). For the development of a TBB, Prediction Analysis ofMicroarrays (PAM) was used to create a classifier in a training set(containing ⅔ of the data, n=29), with subsequent validation in a testset (containing ⅓ of the data, n=14). Overall accuracy was assessedafter 50 random partitions. To test for balanced baseline conditions ofsubgroups in the cohort (train and test set), a t-test and One Way ANOVAwas used, or Mann-Whitney Rank Sum Test and Kruskal-Wallis One Way ANOVAon Ranks if required.

Results

The ability to distinguish patients who will improve their functionalstatus from those who will go on to circulatory collapse and requirecardiac transplant or LVAD placement, remains an important clinicalchallenge.

Patient Characteristics:

A total of 43 EMBs were analyzed with microarray technology to identifyphenotype specific differences in gene expression and to develop aprognostic TBB. Table 1 contains the baseline conditions of allpatients. The table was divided into 4 subgroups according to clinicaloutcome and subdivisions used for PAM analysis, in order to excludepossible bias that may be caused by unbalanced risk parameters in thetrain and test cohorts. There were no significant differences in age,gender, ventricular function, hemodynamics or drug therapy between thesubgroups. The overall population with IDCM presented at an average ageof 46±15 years, with slight overrepresentation of the male gender (67%).All subgroups were at an advanced NYHA stage of 2.6±0.7 with severelycompromised ejection fraction (EF) of 23±13%, average LVIDD of 6.1-1.5cm and pulmonary capillary wedge pressure (PCWP) of 15±9 mmHg.

TABLE 1 Good Poor prognosis prognosis (n = 25) (n = 18) Age 46 ± 15 48 ±17 Male, n (%) 17 (68%) 12 (67%) NYHA, n (%) I 1 (4%) 1 (5%) II 13 (52%)6 (33%) III 10 (40%) 8 (44%) IV 1 (4%) 4 (22%) LVEF, % 24 ± 13 23 ± 13LVIDD, cm 6.4 ± 1   6.3 ± 2   PAP, mmHg Systolic 36 ± 13 41 ± 13Diastolic 16 ± 6  20 ± 11 Pulmonary capillary wedge 13 ± 7  18 ± 10pressure, mmHg Medications, n (%) 8-Antagonist 17 (68%) 13 (72%) ACEinhibitor 17 (68%) 12 (67%) Aldosterone antagonist 4 (16%) 4 (22%)Diuretic 17 (68%) 15 (83%) Intravenous inotropic 0 0 therapy

Microarray Analysis:

An average of 568±92 ng of total RNA was isolated from all EMBs andtested with the Agilent 2100 Bioanalyzer, revealing high integrity andpurity of RNA of all samples with consistent bands of 18S and 28S RNA(FIG. 1). There were 46 significantly over expressed genes in patientswho recovered from heart failure (q<5%, FC>1.2, Table 1) as determinedwith SAM. PAM was used to evaluate the predictive value of this set ofgenes. To achieve this and test for validity, patients were allocatedinto training sets consisting of ⅔ of samples (n=29), and test setscontaining ⅓ of samples (n=14; FIG. 2). This approach resulted in a‘nearest shrunken centroid’ of 45 genes, which distinguished with highaccuracy between high risk patients and those with an excellentprognosis.

TABLE 2 46 significantly overexpressed genes in heart failure patientswith good prognostic outcome (q < 5%, FC > 1.2). affy ID Gene title Foldchange 232669_at Hypoxia inducible factor 3, alpha subunit 1.8 214951_atsolute carrier family 26, member 10 1.8 243482_at Epidermal growthfactor receptor pathway 1.8 substrate 15-like 1 226210_s_at maternallyexpressed 3 1.7 232159_at Epidermal growth factor receptor pathway 1.7substrate 15-like 1 233026_s_at PDZ domain containing 2 1.6 211996_s_atKIAA0220-like protein hypothetical gene 1.6 LOC 283846 243774_at mucin20, cell surface associated 1.6 242551_at Chromosome 18 open readingframe 1 1.6 244548_at Rho GTPase activating protein 26 1.6 244208_atCheckpoint suppressor 1 1.6 239984_at Sodium channel, voltage-gated,type VII, 1.6 alpha 230683_at CDNA: FLJ20892 fis, clone ADKA03430 1.5241869_at apolipoprotein L, 6 1.5 241597_at Arginine-glutamic aciddipeptide 1.5 (RE) repeats 235887_at Smg-6 homolog, nonsense mediated1.5 mRNA decay factor (C. elegans) 229957_at transmembrane protein 911.5 223546_x_at LUC7L-like (S. cerevisiae) 1.5 239567_at Rho GTPaseactivating protein 10 1.5 242194_at Cullin 4A 1.5 1558525_athypothetical protein LOC283901 1.4 227178_at CUG triplet repeat, RNAbinding protein 2 1.4 228198_s_at Mitochondrial ribosomal protein S9 1.4202379_s_at natural killer-tumor recognition sequence 1.4 224260_at CDNAclone 1.4 238643_at Neuroblastoma, suppression of 1.4 tumorigenicity 1232253_at RAD50 homolog (S. cerevisiae) 1.4 227968_at Parkinson diseaese7 domain containing 1 1.4 233197_at kelch-like 9 (Drosophila) 1.4244512_at Transcribed locus, strongly similar to 1.4 XP_001081342.1233443_at hypothetical protein LOC389362 1.4 231275_at FLJ42875 protein1.4 226419_s_at hypothetical protein LOC645460 1.4 201221_s_at smallnuclear ribonuleoprotein 70 kDa 1.4 polypeptide 209354_at tumor necrosisfactor receptor 1.4 family member 14 226571_s_at protein tyrosinephosphatase receptor 1.4 type, S 220728_at EST 1.3 203071_at semadomain, immunoglobulin domain 1.3 (Ig), short basic domain 213946_s_atobscurin-like 1, similar to titin 1.3 isoform N2-B 201394_s_at RNAbinding motif protein 5 1.3 203748_x_at RNA binding motif, singlestranded 1.3 interacting protein 1 223147_s_at WD repeat domain 33 1.3213773_x_at NOL1/NOP2/Sun domain family, 1.3 member 5 1560049_at CUGtriplet repeat, RNA binding protein 2 1.3 243974_at CDNA clone IMAGE:4821815 1.2 201510_at E74-like factor 3(ets domain transcription 1.2factor, epithelial specific)

Validation:

To obtain the overall performance of our biomarker, 50 random partitionswere performed into train and test sets, revealing an overallsensitivity of 74% (95% CI: 69%-79%) and an overall specificity of 90%(95% CI: 87%-93%). The positive predictive value was 85% (95% CI:80%-89%), while the negative predictive value was 82% (95% CI: 78%-86%).The log odds ratio was 3.3. To assess the impact of various hostfactors, the 43 IDCM subjects were divided into multiple two-way stratabased on age (≥ or <50 years), ejection fraction (≥ or <15%), and use ofintravenous inotropes to test if the accuracy of the prognostic markerwas affected by baseline parameters. The sensitivity and specificity ofthe TBB was then assessed, by determining the proportion of correctlyclassified subjects within each stratum. Notably, the predictiveaccuracy was perfect (sensitivity and specificity: 100%) both inpatients over 50 years and patients with ejection fraction over 15%.None of our patients received inotropic medication.

The molecular signature is illustrated in a heatmap, which was createdby Euclidean distance (FIG. 3). This independent approach ofunsupervised clustering additionally confirmed the robustness of thediscovered set of genes with very clear distinction of samples with BPfrom samples with GP. Pathways with major involvement were ion transportmechanisms (13%), neuromuscular development (10%), protein binding (15%)and transcription (26%) www.geneontology.org, FIG. 4).

Prediction of Recovery in Left-Ventricular Function:

Improved clinical outcome is often associated with recovery in LVfunction. Accordingly, the hypothesis that the GP signature would beassociated with improved ejection fraction was tested. Among the studysample (n=43), all subjects in whom paired echocardiography data frombaseline and follow-up was available (n=17) were selected andcharacterized as GP or PP according to their TBB (FIG. 5). Patientsclassified as having GP experienced a substantial improvement in EF from23±13% to 42±5% (P=0.0009), while patients with a PP molecular signaturecontinued to have a depressed EF at 20±3% and 21±3% at baseline andfollow-up, respectively.

DISCUSSION

One of the most valuable applications of genomic information has provento be clinical prediction. Whereas the pattern of differentiallyexpressed genes provides insight into disease etiology, it has also beenused for the development of biomarkers. This approach has been highlysuccessful in neoplastic disease and is emerging in a variety of otherdisease processes. Here, we sought to identify a TBB that predicted theclinical outcome of patients with new onset heart failure. We havedeveloped a highly accurate prognostic biomarker, which is able todistinguish patients with very poor trajectory from those with excellentlong-term prognosis. Importantly this study used biopsies from patientspresenting at an early disease stage, without additional amplificationof RNA. For two main reasons, this approach provides better accuracy andmore representative results compared to previous studies: First, we arereporting initial transcriptomic differences without “noise” ofunspecific compensatory changes that may be activated during the diseaseprogress. Second, we avoided amplification bias which can occur due todifferent binding preferences of primers.

In addition to providing a novel and useful clinical biomarker, thisstudy has offered insight into individual differences in gene expressionthat may be mechanistically involved in a poor outcome from heartfailure. Most notably, we addressed a major unmet need in the managementof heart failure, the ability to accurately assess patient prognosis.While there are emerging biomarkers of prognostic value, these markersdo not dichotomize substantially between patients with very variableoutcomes. Patients with near identical features at presentation,receiving identical therapy, can have dramatically differing prognoses.While some patients undergo complete recovery of their heart functionwithin an average of 5 years, others progress into circulatory collapsewithin the first 2 years of presentation and require aggressivetherapeutic interventions, such as mechanical circulatory assistance orcardiac transplantation.

The developed TBB, containing a nearest shrunken centroid of 45 genes,predicted the phenotype of samples with very high specificity (90%)suggesting its utility in particular as a rule out test and can be usedin addition to current standard risk assessment. Furthermore, results ofour TBB may be included in the assessment of a priority score intransplant lists. In combination with clinical data and laboratoryvalues, our TBB is promising to provide better guidance in patientmanagement.

It is of notice that our prognostic TBB performed perfectly in patientswho were over 50 years old or who had an EF higher than 15%. In thisregard, markers specific for age and function might be considered in thefuture. Future studies will be required to validate the precise clinicalvalue of the TBB approach for new onset HF. Furthermore, our datastrongly indicate a molecular component that impacts the recovery ofpatients with heart failure. Of the 46 differentially expresses genes(Table 2), all were overexpressed in patients who recovered from heartfailure. Many overexpressed genes in these patients have RNA or DNAbinding functions, e.g. RBMS1 and WDR33, playing a crucial role in DNAreplication, gene transcription, cell cycle progression and apoptosisVarious genes with critical regulatory functions were identified, i.e.:SNRP70; the nuclear transcription factors ELF3, CHES1, and RERE; NSUN4,a gene with methyltransferase activity, and the transcription factorHIF3A. HIF3A has been discussed as an inductor of glucose transporters,vascular endothelial growth factor and erythropoietin similar to HIF1Aand HIF2A, while others postulated its counteraction with the other twosubunits.

The genes CUGBP2, LUC7-like and SEMA3B are involved in neuromusculardevelopment, axon guidance and regulation of heart contractility(www.geneontology.org/). Closely related in its function is also theoverexpressed obscurin-like 1 gene, a linker that stabilizes cellcontacts and organelles within the cytoskeleton and which is located atthe intercalated discs in the adult cardiomyocyte. Obscurin-like 1 geneis supposed to have similar functions to obscurin, a multifunctionalprotein responsible for assembly of myofibrils and myocyte cellularorganization. By its interaction with titin and ankyrin, as well aslinking sarcomere and sarcoplasmic reticulum, it provides the requiredalignments for contraction.

Further we discovered two regenerative genes, Rad50 and SMG6, importantkey regulators in telomerase activity and DNA repair. Rad50 is part ofthe Mrel l/Rad50/Nbsl (MRN) complex, a functional unit that generatest-loops by inserting 3′ G-overhangs at human telomere ends. Theset-loops prevent chromosome ends from being recognized as damaged DNA andprovide a template for telomerase and preservation of genome stability.The increase in telomerase activity might explain a protective effectagainst degenerative processes and aging in the heart of patients withgood prognosis. It is attractive to speculate that the reason for betterrecovery may be due to increased cellular regenerative capacity sinceRad50 may be important for viability of stem cells. Several genes withinour prognostic TBB (SNRP 70 kDa, Obscurin like 1 and RNA binding motif)were consistent with findings of a study of 199 human LVAD samples thatindicated that these genes are involved in recovery. There was nooverlap with results published by Lowes et al. (N Engl J Med 2002 May 2;346(18):1357-65; Lowes B D, et al. J Heart Lung Transplant 2006 May;25(5):579-88), who investigated genes overexpressed during recovery fromHF. This may indicate that there are important differences between geneexpression changes caused by therapy and characteristics of geneexpression which may predict clinical outcome.

Several technical features of this study warrant mention. While mostprevious studies have used cardiac tissues obtained at advanced stagesof cardiomyopathy (often at transplantation), we investigated biopsiesfrom patients obtained at first presentation with heart failure.Therefore, the obtained biomarkers are very relevant for a possibleclinical application and suggest initial pathologic changes in thetranscriptome, which may be causative for the disease and possibletherapeutic targets. Additionally, we used a highly efficient RNAprocessing technique, allowing microarray analysis without additionalamplification, and thus avoiding possible bias due to different bindingpreferences of primers and reduction of time and costs with regards to alater clinical application.

Although endomyocardial biopsy is a low risk procedure, alternativemethods for obtaining transcriptomic biomarkers can be developed. Therehas been evidence that affected tissue and circulating blood cells sharea high percentage of common genes. Easy accessibility of PBMCs by asimple venous puncture makes those cells very attractive for a clinicalapplication.

In summary, we have developed a novel transcriptomic biomarker forprognosis in heart failure obtainable from a single endomyocardialbiopsy with potential for a direct clinical application. This approachshould improve individualization of cardiac care and help identifypatients at highest risk for circulatory collapse within the first yearsof presentation with heart failure. The involvement of genes inneuromuscular development, angiogenesis and DNA repair mechanismincluding telomerase activity within the TBB for patients with excellentclinical outcome offers biological plausibility regarding an advantagein recovery.

Example 2: Transcriptomic Biomarker for Predicting Clinical Outcome andPrognosis in Heart Failure Using a Transcriptomic Biomarker

To identify this biomarker (gene signature) heart samples were collectedfrom patients undergoing heart biopsy early in their clinical course.Following this the samples were stored in a biorepository for 5-10 yearsduring which time the outcome of patients were determined.

Endomyocardial biopsy samples from patients with idiopathiccardiomyopathy have been collected at the Johns Hopkins Hospital between1997-2004 and stored in liquid nitrogen. Biopsy samples from patientswith idiopathic cardiomyopathy were chosen from this biorepsitory—18patients with good prognosis and 12 patients with bad prognosis. Badprognosis was defined as occurrence of death or an intervention, namelyleft-ventricular assist device placement or cardiac transplant, in thefirst 2 years after diagnosis, while good prognosis was defined as eventfree survival for greater than 5 years after diagnosis. The MIAME(Minimum information about a microarray experiment) guidelines werefollowed for all experiments. The tissue was homogenized with the MM301instrument from Retsch and total RNA was isolated with the Micro-to-MidiTotal RNA purification system from Invitrogen. Microarray analysis oftotal RNA was performed with the Human Genome U133 Plus 2.0 Array fromAffymetrix. In all samples, both RNA isolation and microarrayhybridization met all indices of quality control as specified in theAffymetrix Guideline for Assessing Sample and Array Quality.

Raw expression values of all microarray chips were preprocessed withRobust Multiarray Average (RMA) in R. In order to find a transcriptomicbiomarker for class prediction of either bad or good prognosis,Prediction Analysis of Microarrays (PAM) implemented in R was used. PAMuses the “nearest shrunken centroid” method for class prediction. Aftertraining of the classifier (supervised clustering) in a subset of 13samples of patients with good prognosis and 7 samples of patients withbad prognosis, a cluster of 9 genes that predicted clinical outcome with60% sensitivity and 100% specificity (FIG. 6, FIG. 10, Table 3, Table 5)was found. The same 9 genes were used to produce a heatmap (unsupervisedclustering), standardized by mean levels of expression (FIG. 8). Eachrow represented one of the 9 genes, and each column was a patientsample. A red cell depicts an underexpressed gene in a given patientrelative to the average gene expression in all patients, while a bluecell denotes an overexpressed gene. The dendrogram at the top is anunsupervised hierarchical clustering algorithm that divides samples intogroups based on the similarity of the gene expression profiles. The“good prognosis samples” and the “poor prognosis samples” segregate intotwo dominant clusters. However, 3 samples were misclassified with thisapproach (sensitivity 75%, specificity 100%). In all cases themisclassifications were bad prognosis patients being misclassified asgood prognosis. The number of genes were increased to 43, in order tominimize misclassification error (FIG. 6, FIG. 7) and developed aclassifier in PAM on the same training set (n=20), which predicted againwith 60% sensitivity and 100% specificity. However, the individualprobabilities for correct class prediction ameliorated slightly, asdenoted in Table 5. With a heatmap including those 43 genes, a higheraccuracy than with PAM was achieved, misclassifying only one sample in30 total (92% sensitivity and 100% specificity). One sample (BP-10) wasmisclassified in all approaches, presumably an outlier (FIG. 9). Thefact that the discovered set of genes performed very accurately on allsamples, strengthens the results of the biomarker signature. Of allgenes contributing to the 9 genes transcriptomic biomarker, only one wassignificantly upregulated in the group with bad prognosis. The set of 43genes contained one additional upregulated unidentified transcript inthe group with bad prognosis (FIG. 11). Tables 6 and 7 list all genescomprised within the 9 and 43 genes signature and that wereoverexpressed in the group of patients with long term survival. The 43gene biomarker contained genes that are of prognostic relevance, as forexample Myosin heavy polypeptide 7. Its mutations have been shown to beassociated with severity of heart failure, in particular in familialdilated cardiomyopathy. The outliers can be reduced by training theclassifier on a higher number of investigated samples. The presentedbiomarker achieved significantly higher prognostic accuracy than classicrisk factors and schemes that have been suggested by other groups, e.g.Seattle Heart Failure Index.

TABLE 3 Prediction for Threshold = 0 Settings Name: Settings4 OffsetQuantile 50 Offset Value 0.3 both RNG Seed 420 Prior Distribution(Sample Prior) Class 1 2 Prob. 0.35 0.65 Test set Prediction ConfusionMatrix True\Predicted 1 2 1 3 2 2 0 5 Actual, Predicted Classes andPredicted Posterior Probabilities Sample BP-10 BP-18 BP-6 BP-7 BP-9GP-20 GP-22 GP-23 GP-24 GP-25 Labels Class 1 1 1 1 1 2 2 2 2 2 LabelsPredicted 2 2 1 1 1 2 2 2 2 2 Bad 0.00842577 0.08808878 0.806138 0.735120.999652 0.000469 0.098695 0.0002 0.000244 0.00194 prognosis Good0.99157422 0.91191122 0.193861 0.26488 0.000348 0.999531 0.901305 0.99980.999756 0.99805 prognosis

Prediction Result of the Test Set (n=10):

9 genes signature was used (Table 3). The sample labels are listed inthe fifth row from below, followed by the actual class labels and thepredicted classes. Bad prognosis (BP) samples were assigned to class1—good prognosis (GP) samples were assigned to class 2. 8 samples werecorrectly classified (probabilities between 73 and 99%). Only twosamples were misclassified (with a probability higher than 90%).Predicted probabilities are listed for each class in the last two lines.

TABLE 4 Prediction for Threshold = 0 Settings Name: Settings5 OffsetQuantile 50 Offset Value 0.350764 both RNG Seed 420473 PriorDistribution (Sample Prior) Class 1 2 Prob. 0.35 0.65 Test setPrediction Confusion Matrix (Thresthold = 0) True\Predicted 1 2 1 3 2 20 5 Actual, Predicted Classes and Predicted Posterior ProbabilitiesSample BP-18 BP-10 BP-6 BP-7 BP-9 GP-20 GP-22 GP-23 GP-24 GP-25 LabelsClass 1 1 1 1 1 2 2 2 2 2 Labels Predicted 2 2 1 1 1 2 2 2 2 2 ClassLabels Bad 0.011971912 1.92842E−06 0.995824315 0.896715 1 2.3E−131.47E−05 2.74E−13 1.48E−12 3.69E−08 prognosis Good 0.9880280880.999998072 0.004175685 0.103285 4.51E−08 1 0.999985 1 1 1 prognosis

Prediction Result of the Test Set (n=10):

43 genes signature (Table 4). Description of the table is the same as intable 6. The probabilities for the right classification increasedlightly.

TABLE 5 Sample BP-10 BP-18 BP-6 BP-7 BP-9 GP-20 GP-22 GP-23 GP-24 GP-25Labels 9 genes signature Bad 0.008425774 0.08808878 0.806138017 0.735120.999652 0.000469 0.098695 0.0002 0.000244 0.001943 prognosis Good0.991574226 0.91191122 0.193861983 0.26488 0.000348 0.999531 0.9013050.9998 0.999756 0.998057 prognosis 43 genes signature Bad 0.0119719121.92842E−06 0.995824315 0.896715 1 2.3E−13 1.47E−05 2.74E−3 1.48E−123.69E−08 prognosis Good 0.988028088  0.999998072 0.004175685 0.1032854.51E−08 1 0.999985 1 1 1 prognosis

Table 5 shows the results from PAM: performance of both 9 genessignature and 43 genes signature. Each sample labeled BP was derivedfrom a patient with bad prognosis, each sample labeled with GP from apatient with good prognosis. The predicted probability for each classusing the developed classifier is listed in each row. Probabilities forcorrect classification increased slightly with increasing the number ofgenes.

TABLE 6 Method used Sensitivity Specificity PAM: 9 genes 60% 100% PAM:43 genes 60% 100% Heatmap: 9 genes 75% 100% Heatmap: 43 genes 92% 100%

Table 6 is an overview of performance of all applied algorithms. Everymethod used was able to predict poor prognostic outcome with 100%specificity. However, unsupervised clustering (heatmap) predicted withhigher sensitivity in general and performed better, especially whenincreasing the number of genes in the molecular signature from 9 to 43genes.

TABLE 7 Probe Gene Title Gene Symbol GO Biological Process Description201655 heparan sulfate proteoglycan 2 (perlecan) HSPG2 cell adhesion,protein localization 208978 cysteine-rich protein 2 CRIP2 — 209354 tumornecrosis factor receptor superfamily, member 14 TNFRSF14 apoptosis,immune response, cell surface receptor linked signal 211996KIAA0220-like protein, nuclear pore complex LOC23117 biological_process213946 obscurin-like 1, similar to titin isoform N2-B OBSL1 — 214951solute carrier family 26, member 10 SLC26A10 transport, regulation ofRho protein signal transduction 227968 Parkinson disease 7 domaincontaining 1 PDDC1 — 241597 Arginine-glutamic acid dipeptide (RE)repeats RERE chromatin remodeling, transcription, NLS-bearing substrateimport 242551 Chromosome 18 open reading frame 1 C18orf1biological_process

Table 7 shows 8 upregulated genes in patients with good prognosis: firstcolumn contains the IDs from Affymetrix for all upregulated transcriptsof the 9 genes biomarker. Only 1 gene was downregulated, namely themuscarinic acetylcholine receptor M3. Annotations for biologicalfunction were obtained from the Gene Ontology website (GO).

TABLE 8 Probe Gene Title Gene Symbol GO Biological Process Description155845 Hypothetical LOC401320 LOC401320 — 156004 CUG triplet repeat, RNAbinding protein 2 CUGBP2 RNA processing, neuromuscular junctiondevelopment, regulation of 201394 RNA binding motif protein 5 RBM5 RNAprocessing, cell cycle, negative regulation of progression 201655heparan sulfate proteoglycan 2 (perlecan) HSPG2 cell adhesion, proteinlocalization 202379 natural killer-tumor recognition sequence NKTRprotein folding 202808 — — — 203071 sema domain, immunoglobulin domain(Ig), short SEMA3B cell-cell signaling, development, axon guidance basic203748 RNA binding motif, single stranded interacting RBMS1 DNAreplication, RNA processing, regulation of translation protein 1 203981ATP-binding cassette, sub-family D (ALD), ABCD4 transport member 4204737 myosin, heavy chain 6, myosin, heavy chain 7 MYH6 /// MYH7striated muscle contraction, muscle contraction 204978 splicing factor,arginine/serine-rich 16 SFRS16 mRNA processing, RNA splicing 206209carbonic anhydrase IV CA4 one-carbon compound metabolism, visualperception, response to 207541 exosome component 10 EXOSC10 mRNAcatabolism, nonsense-mediated decay, rRNA processing 207798 ataxin2-like ATXN2L biological_process 208978 cysteine-rich protein 2 CRIP2 —209354 tumor necrosis factor receptor superfamily, TNFRSF14 apoptosis,immune response, cell surface receptor linked signal 210628 member 14LTBP4 regulation of cell growth, protein folding, development,regulation of latent transforming growth factor beta binding 211909protein 4 PTGER3 transcription, DNA-dependent, signal transduction,G-protein coupled prostaglandin E receptor 3 (subtype EP3) 211996KIAA0220-like protein, nuclear pore complex LOC23117 biological_process212487 G patch domain containing 8 GPATCH8 biological_process 213946obscurin-like 1, similar to titin isoform N2-B OBSL1 — 214951 solutecarrier family 26, member 10 SLC26A10 transport, regulation of Rhoprotein signal transduction 220219 leucine rich repeat containing 37ALRRC37A — 221071 — — — 221780 DEAD (Asp-Glu-Ala-Asp) box polypeptide 27DDX27 — 221806 SET domain containing 5 SETD5 — 221833 Lon peptidase 2,peroxisomal LONP2 proteolysis, ATP-dependent proteolysis 223546LUC7-like (S. cerevisiae) LUC7L negative regulation of striated muscledevelopment 224260 CDNA clone IMAGE: 4478733 — — 225562 RAS p21 proteinactivator 3 RASA3 intracellular signaling cascade, regulation of smallGTPase mediated 226040 MRNA; cDNA DKFZp762Nl56 (from clone — — 227968Parkinson disease 7 domain containing 1 PDDC1 — 228198 Mitochondrialribosomal protein S9 MRPS9 protein biosynthesis, DNA damage response,detection of DNA 229830 Transcribed locus — — 230683 CDNA: FLJ20892 fis,clone ADKA03430 — — 238185 RNA binding motif, single strandedinteracting RBMS1 DNA replication, RNA processing /// regulation oftranslation protein 1 241597 Arginine-glutamic acid dipeptide (RE)repeats RERE chromatin remodeling, transcription, NLS-bearing substrateimport into 242551 Chromosome 18 open reading frame 1 C18orf1biological_process 244208 Checkpoint suppressor 1 CHES1 DNA damagecheckpoint, G2 phase of mitotic cell cycle 244494 Zinc finger, DHHC-typecontaining 1 ZDHHC1 biological_process, protein palmitoylation 244548Rho GTPase activating protein 26 ARHGAP26 signal transduction, nervoussystem development, actin cytoskeleton

Table 8: 41 upregulated genes in patients with good prognosis: 41transcripts of the 43 genes biomarker were overexpressed in the groupwith good clinical outcome. Only 2 genes were downregulated—again themuscarinic acetylcholine receptor M3, as in the 9 genes molecularsignature, and a transcript with unknown function.

Although the invention has been illustrated and described with respectto one or more implementations, equivalent alterations and modificationswill occur to others skilled in the art upon the reading andunderstanding of this specification and the annexed drawings. Inaddition, while a particular feature of the invention may have beendisclosed with respect to only one of several implementations, suchfeature may be combined with one or more other features of the otherimplementations as may be desired and advantageous for any given orparticular application.

The Abstract of the disclosure will allow the reader to quicklyascertain the nature of the technical disclosure. It is submitted withthe understanding that it will not be used to interpret or limit thescope or meaning of the following claims.

1-55. (canceled)
 56. A composition comprising a set of labeled nucleicacid molecules derived from a patient with heart failure wherein thelabeled nucleic acid molecules comprises nucleic acid molecules thatcorrespond to HSPG2, CRIP2, TNFRSF14, LOC23117, OBSL1, SLC26A10, PDDC1,RERE, C18orf1, LOC401320, CUGBP2, RBM5, NKTR, SEMA3B, RBMS1, ABCD4,MYH6, MYH7, SFRS16, CA4, EXOSC10, ATXN2L, LTBP4, PTGER3, GPATCH8,LRRC37A, DDX27, SETD5, LONP2, LUC7L, RASA3, MRPS9, RBMS1, CHES1, ZDHHC1,and ARHGAP26.
 57. The composition of claim 56, wherein the labelednucleic acid molecules are hybridized with a microarray.
 58. Thecomposition of claim 56, wherein the labeled nucleic acid molecules isamplified nucleic acid molecules.
 59. The composition of claim 58,wherein the amplified nucleic acid molecules is amplified by RT-PCR. 60.The composition of claim 56, wherein the labeled nucleic acid moleculesindependently hybridize to sequences that correspond to HSPG2, CRIP2,TNFRSF14, LOC23117, OBSL1, SLC26A10, PDDC1, RERE, C18orf1, LOC401320,CUGBP2, RBM5, NKTR, SEMA3B, RBMS1, ABCD4, MYH6, MYH7, SFRS16, CA4,EXOSC10, ATXN2L, LTBP4, PTGER3, GPATCH8, LRRC37A, DDX27, SETD5, LONP2,LUC7L, RASA3, MRPS9, RBMS1, CHES1, ZDHHC1, and ARHGAP26.
 61. Thecomposition of claim 56, wherein the labeled nucleic acid molecules arelabeled DNA.
 62. The composition of claim 56, wherein the labelednucleic acid molecules are labeled cDNA.
 63. The composition of claim56, wherein the labeled nucleic acid molecules consists of nucleic acidmolecules that correspond to HSPG2, CRIP2, TNFRSF14, LOC23117, OBSL1,SLC26A10, PDDC1, RERE, C18orf1, LOC401320, CUGBP2, RBM5, NKTR, SEMA3B,RBMS1, ABCD4, MYH6, MYH7, SFRS16, CA4, EXOSC10, ATXN2L, LTBP4, PTGER3,GPATCH8, LRRC37A, DDX27, SETD5, LONP2, LUC7L, RASA3, MRPS9, RBMS1,CHES1, ZDHHC1, and ARHGAP26.
 64. A method of generating labeled nucleicacid molecules, the method comprising, labeling nucleic acid moleculesderived from an endomyocardial biopsy sample that correspond to HSPG2,CRIP2, TNFRSF14, LOC23117, OBSL1, SLC26A10, PDDC1, RERE, C18orf1,LOC401320, CUGBP2, RBM5, NKTR, SEMA3B, RBMS1, ABCD4, MYH6, MYH7, SFRS16,CA4, EXOSC10, ATXN2L, LTBP4, PTGER3, GPATCH8, LRRC37A, DDX27, SETD5,LONP2, LUC7L, RASA3, MRPS9, RBMS1, CHES1, ZDHHC1, and ARHGAP26.
 65. Themethod of claim 64, further comprising performing RT-PCR on theendomyocardial biopsy sample prior to labeling the nucleic acidmolecules.
 66. The method of claim 64, wherein the nucleic acidmolecules are labeled with a radioisotope label, a fluorescent label, oran enzyme label.
 67. The method of claim 64, wherein the labeled nucleicacid molecules are label DNA.
 68. The method of claim 64, wherein thelabeled nucleic acid molecules are label cDNA.
 69. The method of claim64, wherein the labeled nucleic acid molecules consists of nucleic acidmolecules that correspond to HSPG2, CRIP2, TNFRSF14, LOC23117, OBSL1,SLC26A10, PDDC1, RERE, C18orf1, LOC401320, CUGBP2, RBM5, NKTR, SEMA3B,RBMS1, ABCD4, MYH6, MYH7, SFRS16, CA4, EXOSC10, ATXN2L, LTBP4, PTGER3,GPATCH8, LRRC37A, DDX27, SETD5, LONP2, LUC7L, RASA3, MRPS9, RBMS1,CHES1, ZDHHC1, and ARHGAP26.